Wiesława Widłak

Tutorial
LNBI 8248

Molecular Biology
Not Only for Bioinformaticians

123
Lecture Notes in Bioinformatics 8248
Edited by S. Istrail, P. Pevzner, and M. Waterman

Editorial Board: A. Apostolico S. Brunak M. Gelfand

T. Lengauer S. Miyano G. Myers M.-F. Sagot D. Sankoff

R. Shamir T. Speed M. Vingron W. Wong

Subseries of Lecture Notes in Computer Science

Wiesława Widłak

Molecular Biology
Not Only for Bioinformaticians

13
Author
Wiesława Widłak
Maria Skłodowska-Curie Memorial Cancer Center
and Institute of Oncology, Gliwice Branch
Wybrzeże Armii Krajowej 15, 44-101 Gliwice, Poland
E-mail: wwidlak@io.gliwice.pl

ISSN 0302-9743 e-ISSN 1611-3349

ISBN 978-3-642-45360-1 e-ISBN 978-3-642-45361-8
DOI 10.1007/978-3-642-45361-8
Springer Heidelberg New York Dordrecht London

Library of Congress Control Number: 2013955613

CR Subject Classification (1998): J.3

LNCS Sublibrary: SL 8 – Bioinformatics

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Preface

The book is addressed to beginners in molecular biology, especially to com-

puter scientists who would like to work as bioinformaticians (computational
biologists). Bioinformatics, which could be defined as the application of com-
puter science and information technology to the field of biology and medicine,
has been rapidly developing over the past few decades. It generates new
knowledge as well as the computational tools to create that knowledge. Cer-
tainly, understanding of the basic processes in living organisms is indispens-
able for bioinformaticians in the creation of knowledge. The book contains
information about these processes presented in a condensed manner. Addi-
tionally, principles of several high-throughput technologies in molecular bi-
ology, which need the assistance of bioinformaticians, are explained from a
biological point of view. Although some processes observed in living organ-
isms are described here in a more detailed fashion, the book may be treated
as an introduction to molecular biology. Thus it could be the first choice for
people who are unfamiliar with the subject.
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

1 Cells and Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 Cell Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 Prokaryotic Cell Structure . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.2 Eukaryotic Cell Structure . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Biological Membranes and Their Lipid Component . . . . . . . . 4
1.2.1 Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.2 Cholesterol and Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.3 Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3.1 Chromosomes and Karyotype . . . . . . . . . . . . . . . . . . . . . 7
1.4 Cytoplasmic Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4.1 Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4.2 Chloroplasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.4.3 Endoplasmic Reticulum and Golgi Complex . . . . . . . . 10
1.4.4 Peroxisomes, Lysosomes, Vacuoles,
and Glyoxysomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.5 Viruses, Bacteriophages, and Virophages . . . . . . . . . . . . . . . . . 11
1.5.1 Classification of Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2 Protein Structure and Function . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.1 Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2 Peptides and Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.3 Levels of Protein Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.3.1 Hydrogen Bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3.2 Secondary Structure of Proteins . . . . . . . . . . . . . . . . . . . 20
2.3.2.1 Alpha Helix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.3.2.2 Beta Strand, Beta Sheet, and Beta Barrel . . . 20
2.3.3 Tertiary and Quaternary Structure of Proteins . . . . . . 21
VIII Contents

2.4 Proteins Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.4.1 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.4.2 Membrane Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.4.2.1 Proteins Involved in Immune Response . . . . . 26
2.4.2.2 Cell Surface Receptors . . . . . . . . . . . . . . . . . . . . 27
2.4.2.3 Ion Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.4.3 Structural Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3 Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.1 Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.1.1 Pentoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.1.2 Nitrogenous Bases of Nucleotides . . . . . . . . . . . . . . . . . . 32
3.2 DNA Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2.1 Nucleotide Chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2.2 DNA Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.3 Structure and Function of RNA . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.3.1 Messenger RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.3.2 Transfer RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.3.3 Ribosomes and rRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.3.4 Non Coding RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.4 The Organization of Genetic Material in the Nucleus -
Chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.4.1 Histones and Nucleosomes . . . . . . . . . . . . . . . . . . . . . . . . 42
3.4.2 Chromatin Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.5 Genetic Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.6 Genes and Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.7 Repetitive Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

4 DNA Replication, Mutations, and Repair . . . . . . . . . . . . . . . . 49

4.1 DNA Replication Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.1.1 DNA Polymerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.1.2 Polymerase Chain Reaction (PCR) . . . . . . . . . . . . . . . . 52
4.2 Telomerases and Cellular Senescence . . . . . . . . . . . . . . . . . . . . . 53
4.3 Topoisomerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.4 Mutations and Their Consequences . . . . . . . . . . . . . . . . . . . . . . 56
4.4.1 Substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.4.2 Deletion and Insertion . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.4.3 Exon Skipping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.4.4 The Relationship between Mutations
and Polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.5 Mutation Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4.5.1 Mutations Caused by UV Light . . . . . . . . . . . . . . . . . . . 61
4.5.2 Mutations Caused by Chemical Agents . . . . . . . . . . . . . 62
Contents IX

4.5.3 Mutations Caused by Replication Errors . . . . . . . . . . . 63

4.5.4 DNA Methylation and CpG (CG) Islands . . . . . . . . . . 63
4.6 Mechanisms of DNA Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.6.1 Base Excision Repair (BER) . . . . . . . . . . . . . . . . . . . . . . 66
4.6.2 Nucleotide Excision Repair (NER) . . . . . . . . . . . . . . . . . 67
4.6.3 DNA Mismatch Repair (MMR) . . . . . . . . . . . . . . . . . . . 68
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

5 Transcription and Posttranscriptional Processes . . . . . . . . . 71

5.1 Gene Expression Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
5.2 Gene Transcription and RNA Polymerases . . . . . . . . . . . . . . . . 72
5.2.1 RNA Polymerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
5.3 DNA Regulatory Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
5.3.1 Response Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
5.4 Structural Motifs of Transcription Factors . . . . . . . . . . . . . . . . 77
5.5 Transcription of Protein-Coding Genes . . . . . . . . . . . . . . . . . . . 78
5.6 Transcription of RNA Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5.7 The Role of Transcription Factors and Chromatin
Structure in Regulation of Transcription . . . . . . . . . . . . . . . . . . 81
5.7.1 Mechanism of Gene Bookmarking . . . . . . . . . . . . . . . . . 82
5.7.2 Insulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
5.8 RNA Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.8.1 Capping Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
5.8.2 Polyadenylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
5.8.3 Processing of Pre-rRNA and Pre-tRNA . . . . . . . . . . . . 87
5.8.4 RNA Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
5.8.5 RNA Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
5.9 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

6 Synthesis and Posttranslational Modifications

of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.1 Protein Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.1.1 The Initiation Signal for Protein Synthesis . . . . . . . . . . 93
6.1.2 Role of tRNA in Translation . . . . . . . . . . . . . . . . . . . . . . 94
6.1.3 Process of Protein Synthesis . . . . . . . . . . . . . . . . . . . . . . 96
6.1.3.1 Initiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
6.1.3.2 Elongation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
6.1.3.3 Termination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
6.1.4 Reading Frame Shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
6.1.5 Overlapping Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
6.2 Posttranslational Modifications and Protein Sorting . . . . . . . . 99
6.2.1 Targeting of Proteins to the Rough Endoplasmic
Reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
X Contents

6.2.2 Transport of Proteins into and across the Golgi

Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
6.2.3 Transport of Proteins into Lysosomes . . . . . . . . . . . . . . 103
6.2.4 Nuclear Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
6.2.4.1 Nuclear Localization Signal and Nuclear
Export Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
6.2.4.2 Mechanism of Importins and Exportins
Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
6.3 Protein Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

7 Cell Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

7.1 Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
7.1.1 CDK and Cyclins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
7.1.2 Mechanism of Initiation and Termination of the S
Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
7.2 Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
7.2.1 Initiation of Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
7.2.2 Mechanism of Mitosis Termination . . . . . . . . . . . . . . . . 113
7.3 Meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
7.3.1 Independent Chromosome Segregation . . . . . . . . . . . . . 114
7.4 DNA Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
7.4.1 Homologous Recombination . . . . . . . . . . . . . . . . . . . . . . . 116
7.4.2 Site-specific Recombination . . . . . . . . . . . . . . . . . . . . . . . 117
7.4.3 Transposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

8 Cell Signaling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

8.1 Signaling through Hydrophobic Molecules . . . . . . . . . . . . . . . . 121
8.2 Signaling through Ion Channel Linked Receptors . . . . . . . . . . 123
8.3 Signal Transduction via Receptors Associated with G
Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
8.3.1 G Protein-Coupled Receptors . . . . . . . . . . . . . . . . . . . . . 125
8.3.2 G Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
8.3.3 Effectors and Secondary Messengers . . . . . . . . . . . . . . . 128
8.4 Signal Transduction through Enzyme Linked Receptors . . . . 130
8.4.1 Receptor Tyrosine Kinases . . . . . . . . . . . . . . . . . . . . . . . 130
8.4.2 Receptor Serine/Threonine Kinases . . . . . . . . . . . . . . . . 132
8.5 Intracellular Signaling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . 132
8.5.1 cAMP-dependent Signaling Pathway . . . . . . . . . . . . . . . 132
8.5.2 NF-kappaB Signaling Pathway . . . . . . . . . . . . . . . . . . . . 133
8.5.3 JAK-STAT Signaling Pathway . . . . . . . . . . . . . . . . . . . . 134
8.5.4 MAPK Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . 135
8.6 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Contents XI

9 High-Throughput Technologies in Molecular Biology . . . . 139

9.1 DNA Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
9.1.1 Chain Termination Sequencing . . . . . . . . . . . . . . . . . . . . 139
9.1.2 Full Genome Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . 141
9.2 Analysis of Gene Expression at RNA Level . . . . . . . . . . . . . . . 143
9.2.1 DNA Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
9.2.1.1 Two-Color Microarrays . . . . . . . . . . . . . . . . . . . 145
9.2.1.2 One-Color Microarrays . . . . . . . . . . . . . . . . . . . 146
9.3 Studies of Gene Expression Regulation by Chromatin
Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
9.4 Analysis of Protein Expression . . . . . . . . . . . . . . . . . . . . . . . . . . 149
9.4.1 MALDI Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . 149
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Chapter 1
Cells and Viruses

Cells are the smallest structural component of all known living organisms
capable of self-maintenance and reproduction. Although cells vary greatly
in their appearance or size, their structure is basically similar. Even the
plant and animal cells show a significant degree of similarity in their overall
organization.
There are two types of cells: eukaryotic and prokaryotic. The main dif-
ference between them is the method of genetic material storage: in eukaryotic
cells — in an isolated nucleus, in prokaryotic cells — directly in the cytoplasm
(there is no nucleus). Prokaryotic cells are usually independent (unicellular),
while eukaryotic cells are often found in multicellular organisms.
Eukaryotic organisms are organisms composed of eukaryotic cells. We clas-
sify here: animals, plants, fungi, and protists (including organisms that do not
fit into the remaining groups). A new classification of prokaryotic organisms
distinguishes archaea (formerly known as archaebacteria) and bacteria (for-
merly eubacteria). Both are single cell organisms. Archaea live in extreme
environments. They are divided into three main groups on the basis of liv-
ing environment: extreme thermophiles (live in high-temperature environ-
ments, rich in sulfur compounds and low pH, such as hot springs and geysers),
extreme halophiles (live in environments with high concentrations of NaCl,
such as the Dead Sea), and methanogens (live in anaerobic environments
such as swamps, produce methane and do not tolerate oxygen).

1.1 Cell Structure

All cells contain cytoplasm, cell membrane, and DNA (the carrier of genetic
information). The cytoplasm is the liquid filling of the cell, with high density,
holding all the cell’s internal sub-structures (called the organelles), except for
the nucleus. It contains also insoluble substances (so called cytoplasmic in-
clusions) and free ribosomes. A cell membrane surrounds the cytoplasm and

W. Widłak: Molecular Biology, LNBI 8248, pp. 1–13, 2013.


c Springer-Verlag Berlin Heidelberg 2013
2 1 Cells and Viruses

isolates cell from the environment. The cell membrane is composed of the
phospholipid bilayer, which contains proteins and carbohydrates. The phos-
pholipid bilayer is also the basic component of other biological membranes.
DNA (deoxyribonucleic acid) is the genetic material. Eukaryotic cells possess
several pieces of DNA, located in organelles surrounded by a phospholipid
membrane (in the nucleus and mitochondria). Prokaryotic cells possess a
single DNA molecule, directly in the cytoplasm.

1.1.1 Prokaryotic Cell Structure

A prokaryotic cell is simpler and smaller than an eukaryotic cell. A typical
bacterial cell is 5μm long and 1μm wide. There are also smaller bacteria
(0.5μm in diameter), or bigger. A prokaryotic cell contains DNA, cytoplasm,
plasma membrane, and also some of the following components: cell wall, ca-
psule, slime, flagellum and fimbriae/pili (Fig 1.1). It does not contain organ-
elles. Prokaryotes are always single-cell organisms; however most are capable
of forming stable aggregate communities. When such communities are en-
cased in a stabilizing polymer matrix (“slime”), they may be called “biofilms”.
Bacterial biofilms may be 100 times more resistant to antibiotics than free-
living unicells and may be nearly impossible to remove from surfaces once
they have colonized them.

Fig. 1.1 Generalized structure of the prokaryotic cell

A bacterial cell wall contains a unique structure called peptidoglycan. Ar-

chaea do not have peptidoglycan, which means that they are resistant to
many antibiotics interacting with the cell wall. Due to differences in cell wall
structure and the effect of staining by the Gram dye, bacteria can be di-
vided into two groups: Gram-negative and Gram-positive. The cell wall of
Gram-negative bacteria consists of three layers: the periplasmic space, which
is an open area on the outside of the plasma membrane, then a thin layer
of peptidoglycan, and an outer membrane surrounding the peptidoglycan.
1.1 Cell Structure 3

In the cell wall of Gram-positive bacteria, there is no periplasmic space and

outer membrane, while the peptidoglycan layer is thicker. As a result, they
are more sensitive to the lysozyme (an enzyme that is naturally present e.g.
in saliva or tears and is able to hydrolyze bacteria cell wall), and penicillin
(antibiotic derived from fungi).
Capsule and slime form hydrophilic surrounding of the cell wall in most ba-
cteria. Capsule sticks more closely to the wall than slime. Flagellum is a tail-
like projection that protrudes from the cell body. It facilitates the movement
of mobile bacteria. Fimbriae and pili are structures similar to short hairs.
They attach cells to other cells, which is important when infecting other
organisms.
Spores are small, haploid and unicellular structures used for reproduction.
They can be found in plants, algae, fungi, protozoa and bacteria. Bacteria
spores have thick wall, which allows them to survive in wide range of tem-
peratures, humidity and other unfavorable conditions, which kills vegetative
forms exhibiting normal life activity.
All information essential for the bacteria survival (in the environment
typical for it) are saved in the chromosome (bacterial nucleoid). Bacterial
chromosome is usually large, circular piece of DNA, but sometimes linear
chromosomes and several copies of them occur. Bacteria may have also plas-
mids. These are (usually) circular DNA molecules that are replicated inde-
pendently of the chromosome. Plasmids often contain genes that could be
very useful, for example include information how to use a compound present
in environment. If this compound is in the environment, the bacteria gain
an obvious advantage over other bacteria, which do not have the ability to
decompose that compound. Plasmids can be different in sizes, from tiny to
megaplasmids exceeding the size of the genomes of some bacteria. Plasmids
are natural vectors, by which continuous exchange of genetic information
among bacteria (sometimes even completely unrelated) occurs. For exam-
ple, many antibiotic resistance genes reside on plasmids, facilitating their
transfer. Plasmids serve as important tools in bioengineering, where they are
commonly used to multiply or express particular genes.

1.1.2 Eukaryotic Cell Structure

Eukaryotic cells contain organelles, which are defined as membrane-enclosed
structures (such as nucleus, mitochondria, chloroplasts, endoplasmic retic-
ulum, Golgi apparatus, lysosomes, vacuoles or peroxisomes). Animal cell is
surrounded only by the plasma membrane, while the plant cell has an ad-
ditional layer called the cell wall, which is formed from cellulose and other
polymers (Fig. 1.2). Typical size of eukaryotic cells varies from 5 to 50 μm.
The nucleus is the largest structure in the eukaryotic cell. It is not part of
the cytoplasm, which can be defined as everything that is surrounded by a
4 1 Cells and Viruses

(a)

(b)

Fig. 1.2 A typical eukaryotic cell illustration: (a) animal; (b) plant

cell membrane, excluding the nucleus. The term “cytoplasm” is however often
used in the narrower sense, meaning only the cytosol. Cytosol is the cytop-
lasm after exclusion of all organelles. It is a liquid, water colloid containing
proteins (suspended or dissolved), lipids, fatty acids, free amino acids and
minerals (e.g. calcium, magnesium, sodium). Complex network of protein
fibrils (microfilaments) and microtubules, forming the cytoskeleton (which
allows the cell to maintain its shape and size), is also an important component
of the cytoplasm.

1.2 Biological Membranes and Their Lipid Component

All biomembranes (cell membrane and membranes of all intracellular or-
ganelles) are highly selective permeable barriers, setting out the boundaries
1.2 Biological Membranes and Their Lipid Component 5

of cells and organelles. They are built from lipids and proteins. The lipids ex-
isting in biological membranes there are mainly phospholipids and cholesterol.
Carbohydrates may be attached to proteins and phospholipids present in the
membranes — forming respectively glycoproteins and glycolipids (Fig. 1.3).
The membranes are liquid layered structures: the majority of lipid and pro-
teins molecules can move fast along the membranes. It is worth noting that
the lipids, besides cell membrane building function, can also be used as “fuel
particles”, energy storing molecules and as signaling molecules (ch. 8).

Fig. 1.3 Scheme of a typical biological membrane (lipid-protein bilayer)

1.2.1 Phospholipids
Phospholipid molecule is composed of hydrophilic (with high affinity for wa-
ter) polar group, so-called “head”, and hydrophobic (with low affinity for
water) “tail”. Hydrophobic tail is made up of two fatty acid chains (Fig. 1.4).
Most polar phospholipids are phosphoglycerides that contain glycerol con-
necting the head with the tail. For example phosphatidylcholine, phosphati-
dylserine, phosphatidylethanolamine or phosphatidylinositol belong to this
group. Chains of fatty acids in biological membranes usually contain an even
number of carbon atoms, generally from 14 to 24. There are saturated (the
neighboring carbon atoms are linked by a single bond) or unsaturated fatty

(a) (b)

Fig. 1.4 The chemical structure of phospholipids: (a) general model, (b) structure
of phosphatidylcholine
6 1 Cells and Viruses

acids (when some of the neighboring carbon atoms are connected by a double
bond). The chain length and degree of saturation of fatty acids in lipids has
significant influence on the fluidity of biological membranes.
When phospholipid molecules are placed in water, the hydrophilic heads
are oriented facing the water and the hydrophobic tails avoid water. As result,
the structure called the bilayer sheet, liposome, or micelle may be formed
(Fig. 1.5).

Fig. 1.5 The structures formed by phospholipids in aqueous solutions. The fig-
ure presents a section through the liposome and micelle and a fragment of the
bilayer sheet. Altered from: http://upload.wikimedia.org/wikipedia/commons/
c/c6/Phospholipids_aqueous_solution_structures.svg

1.2.2 Cholesterol and Steroids

Cholesterol does not exist in most prokaryotic cells, whereas it is a common
component of mammalian cell membranes. It fulfills a critical role in the reg-
ulation of membrane fluidity. Also synthesis of steroid hormones is based on

Fig. 1.6 The structure of cholesterol and other important steroids. They are char-
acterized by the presence of four hydrocarbon rings, marked here as A, B, C and
D. Due to the presence of the hydroxyl group (-OH) cholesterol has amphipathic
properties (it has hydrophobic and hydrophilic parts).
1.3 Nucleus 7

cholesterol (Fig. 1.6; ch. 8.1). Accumulation of cholesterol in an artery wall

plays a key role in atherosclerosis — disorder, which can lead to heart attack
or stroke.

1.3 Nucleus
The nucleus consists of a nuclear envelope (constructed from two membranes;
Fig. 1.7), the nucleolus and nucleoplasm. The outer nuclear membrane is con-
tinuous with the membrane of the rough endoplasmic reticulum (RER). The
space between the membranes (perinuclear space) is continuous with the
RER lumen. With the inner face of the nuclear envelope the nuclear lamina
is associated, which is built from fibrous proteins called lamins. The nuclear
lamina binds inner nuclear membrane with genetic material. The genetic
material formed in the chromatin/chromosomes is located mainly in nucleo-
plasm. However, the parts of various chromosomes, which contain groups of
genes encoding rRNA (ch. 3.3.3), could be concentrated in one region forming
the nucleolus. Its main role is the production of rRNA.

Fig. 1.7 Scheme of a nuclear envelope which is built from two layers of double
lipid membrane. In the nucleus of mammals there are about 4,000 nuclear canals
(pores) present, each composed of more than 100 various proteins.

1.3.1 Chromosomes and Karyotype

Chromosome is condensed structure, which is formed from a single DNA
molecule and associated proteins. In cells which are not dividing at the mo-
ment, chromosomes are not visible in light microscope, because DNA together
with accompanying proteins (i.e. chromatin) is loosely spreaded across the
nucleus. During cell division, in the stage of metaphase (see ch. 7), chromatin
condenses and becomes visible in the form of chromosomes.
Each chromosome has the short and long arm (designated as p, from the
“petit” and q, from the “queue”). The arms are separated by narrow region
8 1 Cells and Viruses

called the centromere (Fig. 1.8). The chromosomes of the same shape and size,
containing similar genetic information (i.e. genes) are called homologous chro-
mosomes. In diploid cells, one of them comes from the mother and the other
from the father. During cell division, at metaphase, replicated chromosomes
contain two sister chromatids linked with centromere. Sister chromatids are
identical copies of the chromosome: they contain the same genes and the
same alleles (i.e. versions of genes in a particular location — locus — on
the homologous chromosome). However, homologous chromosomes contain
the same genes, but alleles can vary. During cell division pairs of chromo-
somes are separated into two daughter cells.

Fig. 1.8 Homologous chromosomes during mitotic cell division

Chromosomes are visible under the microscope only after staining. Then,
they look like strings with dark and light transverse stripes. A set of chromo-
somes present in the cell during metaphase is called a karyotype. Human
germ cells (egg or sperm) contain one set of chromosomes (haploid cell, 1n),
that is 23 pieces: 22 autosomes and the X or Y. Somatic cells (remaining cells
of the body) normally contain two homologous sets of chromosomes, i.e. 46
chromosomes (diploid, 2n). One set comes from mother, the other — from
father. In other species the number of chromosomes varies from 1 to 1260.
Human somatic cells contain two chromosomes that determine sex: XX in
females and XY in males. In germ cells there is only one sex chromosome: in
female it is always X chromosome, in male — X or Y.
1.4 Cytoplasmic Organelles 9

1.4 Cytoplasmic Organelles

Organelles can be defined as intracellular, bounded by membrane, protein-
lipid structures (the cell nucleus is also an organellum). In cytoplasm there are
present mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus,
peroxisomes, lysosomes, vacuoles, and glyoxysomes.

1.4.1 Mitochondria
Eukaryotic cells have a lot of mitochondria — they can be up to a quarter of
the cytoplasm volume. They are elongated organelles with a diameter ranging
from 0.5 to 10 μm (the size corresponds to a bacterial cell). Mitochondria are
composed from two membranes: outer and inner, which is strongly folded.
Mitochondria have their own DNA (defined as the mtDNA) coding for pro-
teins and RNA. However the majority of mitochondrial proteins are encoded
by nuclear DNA.

Fig. 1.9 Chemical structure of ATP (adenosine triphosphate)

The main task of mitochondria is the production of ATP (adenosine

triphosphate), which provides source of energy for most cellular processes
(Fig. 1.9). Energy is stored in phosphate bonds and can be released during
the hydrolysis of ATP to ADP (adenosine diphosphate) and AMP (adenosine
monophosphate).
In animal cells, the catabolism (degradation of glucose and fatty acids)
is the main source of energy for ATP synthesis. The production of ATP ta-
kes place in the inner mitochondrial membrane. Energy is transferred from
electron transport chain to the ATP synthase by movements of protons across
this membrane. As a result of disturbances in electron transport, the free ra-
dicals (and other reactive oxygen species) can be generated, which can lead
to damage of cell structures and to cell senescence.
10 1 Cells and Viruses

1.4.2 Chloroplasts
Cloroplasts, like mitochondria, are composed from inner and outer mem-
brane. Chloroplasts also have their own DNA, but most of their proteins are
encoded by nuclear DNA. The inner membrane of the chloroplast forms thy-
lakoids, that contain chlorophyll. It absorbs light necessary for the light re-
action of photosynthesis. In this stage, thanks to the energy absorbed from
light quanta, oxygen molecule and hydrogen ions are formed from two wa-
ter molecules. This results in forming of the concentration gradient in the
thylakoid membrane. The flow of ions through the membrane is connected
with the synthesis of ATP. Stroma is the liquid component of the chloro-
plast, in which, using the energy from ATP, CO2 is assimilated and synthesis
of saccharides occurs (dark reactions of photosynthesis).

1.4.3 Endoplasmic Reticulum and Golgi Complex

Endoplasmic reticulum (ER) forms an irregular network of cisternae, vesicles
and tubules inside the cell isolated from the cytosol by biological membranes.
It exists in two forms: smooth and rough. The main role of the rough endoplas-
mic reticulum is to process the newly synthesized proteins, so it is associated
with ribosomes (ch. 6.2.1). Smooth endoplasmic reticulum is involved in the
synthesis and metabolism of lipids. It appears in highest quantities in liver
cells (hepatocytes).
After the initial processing of proteins in rough endoplasmic reticulum,
they are closed in transport vesicles and directed to other membranous struc-
tures — the Golgi apparatus (ch. 6.2.2). These structures are the main place
of sorting and modifications of proteins and lipids. Here, glycoproteins are
produced and they are transported further to their destinations. Golgi appa-
ratus is also a place of synthesis of polysaccharides and mucopolysaccharides.

1.4.4 Peroxisomes, Lysosomes, Vacuoles, and

Glyoxysomes
Peroxisomes contain enzymes, which allow degradation of amino acids and
fatty acids. In these reactions the hydrogen peroxide is formed, which is ha-
rmful to cells. It is converted to water and oxygen by an enzyme called
catalase.
The main function of lysosomes is degradation of unnecessary macromole-
cules. Macromolecules are hydrolyzed by the respective enzymes: nucleic acids
(DNA and RNA) by nucleases, proteins by proteases, and lipids by lipa-
ses. Lysosomes are present only in animal cells. In plant cells their functions
are performed by the vacuoles.
1.5 Viruses, Bacteriophages, and Virophages 11

Vacuoles store water, ions, saccharides, amino acids and other small mo-
lecules. They can also store unnecessary cell products, which can undergo de-
gradation here. Vacuoles usually fill about 30% of the cell volume, but can
increase its volume up to 90%.
Glyoxysomes are found mainly in plant seeds. Their main function is to
convert fatty acids into acetyl-CoA molecules, which in glyoxylate cycle are
converted into monosaccharides. This mechanism is important in the use of
fat reserves of oilseeds. It occurs during germination and when it is necessary
to launch a non-carbohydrate energy reserves in plants. Glyoxysomes and
peroxisomes are also called microbodies.

1.5 Viruses, Bacteriophages, and Virophages

Viruses are macromolecular complexes built from proteins and nucleic acids.
Viruses are not able to replicate by themselves — so they can not be treated
as living organisms. Replication of viruses is possible only within other cells,
using enzymes from infected cells. Because of that viruses are intracellular
parasites. Most viruses have a diameter between 10 and 300 nm (but disc-
overed in 2013 Pandoraviruses have a size approaching 1μm). The molecule
of the virus (virion) consists of one or more molecules of DNA or RNA (as
a carrier of genetic information of the virus) and a protein shell called a
capsid. Some viruses have an additional lipid envelope which surrounds the
capsid. Most viruses are pathogens — after penetration into living cells they
cause the diseases. Various viruses specifically attack different types of cells
(e.g. the HIV virus attacks only the immune cells, mainly T lymphocytes).
When a virus encounters “his” cell, it attaches to the cell membrane using
specific receptors (proteins on the surface of host cells, which normally have
other functions). Then, it can inject its genetic material into the cell or whole
virus is absorbed entirely by the cell. Inside the cell, based on the information
contained in the genetic material of the virus, there are created new virus
molecules. They can remain in the cell for a long latent phase, or leave after
the cell destruction. It sometimes happens, that the virus remains in the cell
in form of genetic material integrated with the host genome.
Viruses can also infect bacteria. Such viruses are called bacteriophages
(or commonly phages). Bacteriophages can be used for treating bacterial
infections as an alternative to antibiotics. In biotechnology and genetic en-
gineering, phages and other viruses are used to transfer genetic information
between cells (as so-called “vectors”).
There are also viruses that infect other viruses. The first known, called
Sputnik, was discovered in 2008. It is a subviral agent that reproduces
in amoeba cells which are already infected by a certain helper virus.
12 1 Cells and Viruses

Sputnik uses the helper virus’s machinery for reproduction and inhibits repli-
cation of the helper virus. Viruses that depend on co-infection of the host
cell by helper viruses are known as satellite viruses. They act as a para-
site of helper viruses. In analogy to the term bacteriophage they were called
virophages.
The risk associated with viral infections may be very different. Many viral
infections do not cause diseases, for example, most people are infected with
cytomegalovirus (CMV), however, do not show symptoms of the disease.
There are also viruses that often lead to death of the organism, such as coro-
navirus which cause Severe Acute Respiratory Syndrome (SARS) or the HIV
virus causing AIDS.

1.5.1 Classification of Viruses

The classification of viruses could be based on the type of genetic material
held by the virus and on strategy of genetic material replication (the Bal-
timore classification). Besides double-stranded DNA, the carrier of genetic
information in prokaryotic and eukaryotic cells, also single-stranded DNA or
RNA can be a genetic material in viruses. Viruses were divided into seven
classes (ds — double strand, ss — single strand):
I. dsDNA viruses — contain double-stranded DNA;
II. ssDNA viruses — contain single-stranded DNA;
III. dsRNA viruses — contain double-stranded RNA;
IV. ssRNA(+) viruses — contain single-stranded RNA, (+) sense;
V. ssRNA (-) viruses — contain single-stranded RNA, (-) sense;
VI. RNA viruses that use virally encoded reverse transcriptase to
produce DNA from the initial virion RNA genome;
VII. DNA viruses that use virally encoded reverse transcriptase.
The last two classes of viruses are known as retroviruses. Rewriting of the
genetic information from RNA to DNA is an essential part of their replica-
tion cycle. This reaction is catalyzed by reverse transcriptase — an enzyme
encoded by the virus genome (ch. 5.9).

References
1. Adams, M. (ed.): Subcellular Compartments. In: Miko, I. (ed.) Cell Biology. Na-
ture Education (2011), http://www.nature.com/scitable/topic/
subcellular-compartments-14122679
2. Coté, G., De Tullio, M. (eds.): Cell Origins and Metabolism. In: Miko, I. (ed.)
Cell Biology. Nature Education (2011), http://www.nature.com/scitable/
topic/cell-origins-and-metabolism-14122694
3. Davidson, M.W.: Introduction to Cell and Virus Structure. In: Molecular Ex-
pressions Website. The Florida State University (2000),
http://micro.magnet.fsu.edu/cells/index.html
References 13

4. La Scola, B., Desnues, C., Pagnier, I., et al.: The virophage as a unique parasite
of the giant mimivirus. Nature 455, 100–104 (2008)
5. Liang, B.: Construction of the Cell Membrane. Wisc-Online.com (2001),
http://www.wisc-online.com/objects/ViewObject.aspx?ID=AP1101
6. Lodish, H., Berk, A., Zipursky, S.L., et al.: The Dynamic Cell. In: Molecular
Cell Biology, 4th edn., W.H. Freeman, New York (2000)
7. Mc Grath, S., van Sinderen, D. (eds.): Bacteriophage: Genetics and Molecular
Biology, 1st edn. Caister Academic Press, Norfolk (2007)
8. O’Connor, C.M., Adams, J.U.: Essentials of Cell Biology. NPG Education, Cam-
bridge (2010)
9. Ogata, H., Claverie, J.M.: Microbiology. How to Infect a Mimivirus. Science 321,
1305–1306 (2008)
Chapter 2
Protein Structure and Function

Proteins are linear chains of amino acids and are fundamental components
of all living cells (along with carbohydrates, fats and nucleic acids). They
make up half the dry weight of an Escherichia coli (the most widely stud-
ied prokaryotic model organism) cell, whereas other macromolecules such as
DNA and RNA make up only 3% and 20%, respectively. Within cells, as well
as outside, proteins serve a myriad of functions. The chief characteristic of
proteins that allows their diverse set of functions is their ability to bind other
molecules (proteins or small-molecule substrates) specifically and tightly.

2.1 Amino Acids

Amino acids serve as the building blocks of proteins. Amino acid (in short: aa)
is defined as a molecule containing an amine group (-NH2 ), carboxyl group
(-COOH) and the variable group denoted as R, different among different
amino acids (Fig. 2.1). R group is also called the side chain. The overall
amino acid formula can be represented as: R-CH(NH2)-COOH. An average
molecular weight is about 135 daltons.

Fig. 2.1 Schematic diagram of the amino acid: amine group — on the left, car-
boxylic acid group — on the right, R — side chain (variable). The carbon atom
located between the amine group and carboxylic acid group is called α-carbon.

W. Widłak: Molecular Biology, LNBI 8248, pp. 15–29, 2013.


c Springer-Verlag Berlin Heidelberg 2013
16 2 Protein Structure and Function

In aqueous solutions amino acids can be found in the form of inner salts
(they have amphiprotic properties) — they can attach or detach protons to
the functional groups (i.e. amine and carboxylic acid groups). Therefore, the
isoelectric point (pI) is an important criterion, which characterizes amino
acids. pI is at such a pH at which a population of molecules contains approx-
imately the same number of positive as negative charges (the total charge
is zero).
There are known about 300 naturally found amino acids, but only 20 of
them make up the proteins (Fig. 2.2). They are known as proteinogenic
amino acids. They belong to the α-amino acids, in which both functional
groups and side R chain are combined with one carbon atom, called α-carbon.

Fig. 2.2 The names, symbols (three- and one-letter), and the chemical structure
of proteinogenic amino acids

These 20 are encoded by the universal genetic code (ch. 3.5). Nine standard
amino acids are called “essential” for humans because they cannot be created
from other compounds by the human body, and so must be taken in as food.
2.2 Peptides and Proteins 17

Proteinogenic amino acids (except for glycine, which has no asymmetric

carbon atom in the molecule because hydrogen is here the R group), are op-
tically active compounds. Proteinogenic amino acids belong to the group of
L-amino acids, which means that they have an amino group on the left side
of the R chain.
Based on physical and chemical properties of the side chain, amino acids
are divided into:
acidic (aspartic acid and glutamic acid; named also aspartate or gluta-
mate) — in a neutral solution a side chain containing carboxyl group may
lose a proton and the amino acid becomes negatively charged;
basic (lysine, arginine, histidine) — in a neutral solution a side chain con-
taining amino or imino group can gain a proton and become positively char-
ged (the interactions between the positively and negatively charged side chains
of acidic or alkaline amino acids can lead to the formation of “salt bridges”
which stabilize protein structure);
aromatic (tyrosine, tryptophan, phenylalanine) — their side chain con-
tains an aromatic ring;
sulfur (cysteine and methionine) — their side chain contains sulfur (disul-
fide bonds formed between two cysteines strongly stabilize the structure of
the protein, while from methionine the synthesis of all proteins starts);
uncharged hydrophilic (serine, threonine, asparagine, glutamine) —
they have a side chain which contains a hydrophilic (but not undergoing
ionization) functional group, for example the hydroxyl group (such chains
are involved in the formation of hydrogen bonds, stabilizing the protein
structure);
inactive hydrophobic (glycine, alanine, valine, leucine, isoleucine) —
they have a hydrocarbon side chain without functional groups (such chains
can participate in hydrophobic interactions, in protein structure they are
usually directed to the inside);
special structure — in this group is proline, in which the R group is
directly linked to the amine group (the spatial structure of the polypeptide
chain is turned and stiffened in a place of proline).

2.2 Peptides and Proteins

Amino acids can be linked together by peptide bonds in varying sequences
to form a vast variety of peptides or proteins. Peptides are distinguished
from proteins on the basis of size, typically containing less than 50 monomer
units. Oligonucleotides consist of between 2 and 20 amino acids, while
polypeptides refer to the longer peptides (the size boundaries are arbitrary).
18 2 Protein Structure and Function

Proteins consist of one or more polypeptides arranged in a biologically func-

tional way and are often bound to cofactors, or other proteins.
The molecular weight of most polypeptides varies between 5,500 to 220,000.
The mass of the proteins is given in Daltons (named after John Dalton, who
has formulated atomic theory of matter building): one dalton corresponds to
one atomic mass unit. Thus, a protein with a molecular weight of 50,000 has
a weight of 50,000 daltons, or 50 kDa (kilodaltons).
Peptide bond is formed by the condensation reaction. It is an amide
bond formed between the nitrogen from α-amine group of one amino acid
and carbon from α-carboxyl group of another amino acid (Fig. 2.3). Peptide
bond is a flat and rigid structure, while bonds between α-carbon and amine
group or carboxyl group have a possibility to rotate. The line formed by the
repeating peptide bonds is the backbone of the peptide chain.

Fig. 2.3 Formation of the peptide bond by condensation reaction. Chain of amino
acids linked by peptide bonds forms a peptide or a protein.

2.3 Levels of Protein Structure

Structural features of proteins are usually described at four levels of
complexity:
1. Primary structure: the linear arrangement of amino acids (also called a re-
sidue) in a protein and the location of covalent linkages such as disulfide
bonds between amino acids. It is always given starting from the N-termi-
nus (where free α-amine group is present). The C-terminus it is an op-
posite end, with a free carboxyl group (Fig. 2.4). The primary structure
of a protein can readily be deduced from the nucleotide sequence of the
corresponding messenger RNA.
2. Secondary structure: areas of folding or coiling within a protein, which are
stabilized by hydrogen bonding. A polypeptide chain is usually arranged
in the shape of α-helix or β-strand.
3. Tertiary structure: the final three-dimensional structure of a protein, which
results from a large number of non-covalent interactions between amino
acids.
2.3 Levels of Protein Structure 19

Fig. 2.4 Primary structure (sequence of amino acids) of protein on the example
of ribonuclease A (RNase A), the enzyme digesting RNA. Each letter corresponds
to one amino acid (residue).

4. Quaternary structure: non-covalent interactions that bind multiple polypep-

tides into a single, larger protein.

2.3.1 Hydrogen Bonds

Hydrogen bond is a type of relatively weak chemical bond consisting mainly
on electrostatic attraction, but without the ionization of molecules (Fig. 2.5).
A bond is formed by three atoms: one hydrogen and two negatively charged
atoms, usually nitrogen (N) or oxygen (O). The hydrogen atom is connected
by a covalent bond with one of the negatively charged atoms, called the
donor. The second atom is called the acceptor. The electric charge is dis-
tributed between the three atoms that form a bond: electronegative atoms
have partially negative charge, and hydrogen atom has partially positive cha-
rge. The strength of hydrogen bond depends on the donor and acceptor and
their surrounding. Hydrogen bonds are an important factor stabilizing the
structure of macromolecules such as proteins and nucleic acids.

(a) (b) (c)

Fig. 2.5 Hydrogen bonds (denoted by dashed red line): (a) general formula; D:
donor, A: acceptor; (b) the hydrogen bond between two water molecules; (c) the
hydrogen bond in a polypeptide, e.g. α-helix or between two β-chains
20 2 Protein Structure and Function

2.3.2 Secondary Structure of Proteins

2.3.2.1 Alpha Helix
Alpha-helix is formed by amino acid chain in which every 3.6 amino acids ma-
ke one turn. The distance between two turns is 0.54 nm. Shape of α-helix
resembles a cylinder formed by tightly twisted chain (Fig. 2.6). The walls
of the cylinder are created by polypeptide backbone when side chains (subs-
tituents) stick out of the cylinder. The conformation is stabilized by a periodic
hydrogen bonds between amine group and an oxygen atom of the carbonyl
group within the backbone of one polypeptide. α-helix can be right- or left-
handed, although the right-handed form is the common one. If one side of the
helix contains mainly hydrophilic amino acids, and the other — hydrophobic,
α-helix has amphipathic properties (containing both polar and nonpolar por-
tions in the structure). Among types of local structure in proteins, the α-helix
is the most regular and the most prevalent, as well as the most predictable
from sequence.

Fig. 2.6 Scheme of regular α-helix. R — side chains of amino acids Altered from:
http://www.answers.com/topic/alpha-helix

2.3.2.2 Beta Strand, Beta Sheet, and Beta Barrel

Beta-strand is almost fully stretched polypeptide fragment, usually a length
of 5-10 amino acids. R groups of two neighboring amino acids are directed
in the opposite directions. β-sheet is composed from two or more β-strands
(Fig. 2.7). Its structure is stabilized by hydrogen bonds between amine groups
2.3 Levels of Protein Structure 21

and oxygen atoms from carbonyl groups of two neighboring chains (such way
of spatial arrangement of amino acids resembles a piece of paper folded into
a harmonica).

Fig. 2.7 Scheme of β-sheet with two anti-parallel β-strands connected by hydrogen
bonds. Altered from: http://en.wikipedia.org/wiki/
File:BetaPleatedSheetProtein.png

Beta-sheet can have parallel, anti-parallel or mixed arrangement. These

structures (in contrast to the α-helix constructed from one continuous se-
quence) could consist from separate chain fragments, sometimes of different
primary structure. Big β-sheets can form enclosed structures, in which the
first chain connects with the last chain by hydrogen bonds. In this way the
structure of β-barrel is created.
Secondary structure of protein can be predicted on the basis of primary
structure. For this purpose programs available on websites can be used. Some
examples are:
http://bioinf.cs.ucl.ac.uk/psipred/
http://compbio.soe.ucsc.edu/SAM_T08/T08-query.html
http://distill.ucd.ie/porter/
http://www.predictprotein.org/
http://sable.cchmc.org/

2.3.3 Tertiary and Quaternary Structure of Proteins

Tertiary structure describes the spatial organization of protein at a higher
level than the secondary structure. Tertiary structure is the overall spatial dis-
tribution and interrelationships of turned chains and each amino acid residues
in a single polypeptide chain (Fig. 2.8a). It is characterized by high compact-
ness and it has no empty spaces inside of the molecule. Hydrophilic side chains
and/or chains with a charge are typically on the surface of the molecule and
they interact with water molecules. Large hydrophobic groups are generally
hidden inside the protein molecule.
22 2 Protein Structure and Function

(a) (b)

Fig. 2.8 An example of a tertiary (a) and quaternary (b) structure of the protein.
A single peptide with conformation of α-helix and/or β-sheet may additionally fold,
forming a tertiary structure. Quaternary structure is formed by aggregation of two
or more peptides. Sourced from: http://chsweb.lr.k12.nj.us/mstanley/
outlines/organicAP/aporgchem.html

If the protein is composed from several polypeptides (so-called oligomer),

a structure that is formed is called quaternary structure (Fig. 2.8b). The
structure of the proteins is examined mainly by using X-ray crystallography
and nuclear magnetic resonance (NMR).
Tertiary and quaternary structures of proteins are stabilized by different
types of interactions between side chains of amino acids (hydrogen bonds and
ionic bonds, hydrophobic interactions, covalent disulfide bonds).

2.4 Proteins Functions

Proteins are macromolecules with the greatest degree of diversity. It was esti-
mated that in eukaryotic cells dozens-hundreds of thousands of different types
of proteins are present. The structure of these proteins determines the func-
tions they perform in the cell. The most important functions of proteins are:
– enzymatic catalysis — from the hydration of carbon dioxide to chromo-
some replication;
– immune reaction — e.g. immunoglobulins;
– regulatory and signaling (e.g. hormones and growth factors and their re-
ceptors that regulate biochemical processes and signal transduction in cells
and between cells);
– control of membrane permeability — regulation of metabolite concentra-
tions in cell;
– cell adhesion — e.g. catenin and cadherin;
– transport and storage of other molecules — e.g. hemoglobin, transferring,
ovalbumin and casein;
2.4 Proteins Functions 23

– ordered movement (for example muscle contraction) — e.g. actin, myosin;

– building and structural function — e.g. keratin, elastin, collagen.

Beside classification based on function, proteins could be classified by loca-

tion in the living cell. According to this, it is possible to classify all pro-
teins into four main groups: membrane or transmembrane proteins, internal
proteins, external or secretory proteins, and virus proteins. Another clas-
sification, based on posttranslational modifications, split all proteins into
overlapped groups:
– native proteins (not changed after translation),
– glycoproteins (modified by covalent binding with linear or branched
oligosaccharides),
– cleaved proteins (cleaved into two or more pieces),
– proteins with disulphide bonds (pair of cysteins are linked between each
other by disulphide bond, called also disulphide bridge),
– protein complexes,
– chemically modified,
– prions.
Some main protein functions are described below.

2.4.1 Enzymes
Enzymes are proteins that catalyze (accelerate) biochemical reactions in the
cell. It is the best-known role of proteins. Almost all chemical processes in
living organisms (and viruses) require the participation of enzymes to provide
adequate efficiency of the reaction. Enzymes are highly specific to substrates
and, therefore, one enzyme catalyzes only a few reactions from many possible
for given substrates. In this way enzymes determine metabolic and biochem-
ical processes associated with the functioning of living organisms.
Enzymes, like other catalysts, accelerate the reaction by decreasing the
energy barrier (activation energy) between the substrate (S) and product (P)
(Fig. 2.9). The enzyme (E) needs a small portion of energy for the reaction
because it forms transient connection with the substrate (S). This connec-
tion is called the enzyme-substrate complex (ES). The reaction proceeds as
follows:
SUBSTRATE + ENZYME ↔ COMPLEX E-S → PRODUCT + ENZYME

Based on catalyzed reactions, enzymes were divided into following classes:

1. Oxidoreductases (e.g. dehydrogenases, oxidases, reductases, catalases)
— catalyze the oxidation and reduction reactions.
2. Transferases (e.g. acetyltransferases, methylases, kinases) — catalyze
the transfer of functional groups (respectively: acetyl, methyl, phosphate
groups; Fig. 2.10).
24 2 Protein Structure and Function

Fig. 2.9 Reduction of the activation energy in a reaction catalyzed by an enzyme

(a)

(b)

(c)

Fig. 2.10 Examples of the main types of chemical reactions proceeded by en-
zymes: (a) acetylation — addition of an acetyl group, -C(=O)CH3 , catalyzed by
acetyltransferase, e.g. to the side chain of amino acid (in this case — lysine); (b)
methylation — attachment of a methyl group, -CH3 , catalyzed by methylase, e.g. to
cytosine in DNA chain; (c) phosphorylation — attachment of a phosphate group,
-H2 PO4 , catalyzed by protein kinase, e.g. to the tyrosine side chain (serine and
threonine may also be phosphorylated). Substituted groups are marked with red.
2.4 Proteins Functions 25

3. Hydrolases — catalyze hydrolysis reactions in which the molecule is

split into two or more smaller parts with the participation of water. They
include:
• Proteases — digest protein molecules; e.g. caspases which play a
crucial role in apoptosis;
• Nucleases — digest nucleic acids: DNA is digested by DNase, and
RNA by RNase. Exonucleases cut the single nucleotides from the end
of the DNA or RNA molecule, endonucleases cut inside the molecule;
• Phosphatases — catalyze dephosphorylation (removal of a phosphate
group).
4. Lyases (e.g. decarboxylase and aldolase) — catalyze cutting C-C, C-O,
C-S and C-N bonds in a manner other than hydrolysis or oxidation.
5. Isomerases (e.g. epimerases, racemases) — catalyze the atomic rearrange-
ments within the molecule.
6. Ligases (e.g. synthetases, DNA ligase and RNA ligase) — catalyze the
reaction of two molecules joining.

Enzymes can be inhibited by different types of molecules. Some of these

molecules are competitive inhibitors, competing with substrates for reversible
binding to the active center of the enzyme. The non-competitive inhibitors
bind firmly to the enzyme molecules, permanently “removing” part of enzyme
from the system. Enzyme inhibitors are often used as drugs. The best known
enzymes which are targets of therapy are:
• HIV protease — essential for the replication of HIV (the inhibitor is used
in treatment of AIDS);
• angiotensin converting enzyme (ACE) — causes contraction of blood ves-
sels (an ACE inhibitor is used in treatment of high blood pressure);
• HMG-CoA reductase — an essential for the synthesis of cholesterol (its
inhibitors, e.g. statins, reduce cholesterol levels);
• cGMP phosphodiesterase — catalyzes the conversion of cGMP (cyclic
GMP) to GMP (inhibited by Viagra, which is used for the treatment
of erectile dysfunction).

2.4.2 Membrane Proteins

Membrane protein is a protein associated with a cell or an organelle mem-
brane. It was estimated that more than half of all cellular proteins interact
with membranes. Many of them are an integral part of the phospholipid mem-
brane (transmembrane proteins or permanently embedded in the membrane
from one side). The peripheral membrane proteins are transiently associ-
ated with the phospholipid membrane or with integral membrane proteins.
26 2 Protein Structure and Function

Structural proteins existing in the membrane are attached to the microfila-

ments of the cytoskeleton. This provides stability of the cell. Proteins present
on the cell surface allow cells to recognize and interact with each other. These
proteins are involved for example in the immune response (ch. 2.4.2.1). Mem-
brane enzymes produce a variety of substances essential for the cell to func-
tion. Membrane receptors allow the transmission of signals between the cell
and the outside environment (ch. 2.4.2.2). Membrane transport protein play
an important role in maintaining the proper concentration of ions. These
proteins are classified into two groups: the carrier proteins and the channel
proteins (ch. 2.4.2.3).

2.4.2.1 Proteins Involved in Immune Response

An immune system protects an organism against disease. In order to func-
tion properly, it must detect a wide variety of foreign antigens (i.e. substances
that induce an immune response, what results in production of antibodies),
and distinguish them from the organism’s own healthy tissue. In this process,
proteins belonging to the immunoglobulin family are involved. Molecules are
categorized as members of this family based on shared structural features
with immunoglobulins (also known as antibodies); they all possess a domain
known as an immunoglobulin domain. The antibodies produced by B cells
(lymphocytes), present in the peripheral blood, do not have a transmembrane
domain and are soluble secretory proteins. However, many proteins transfer-
ring signals associated with the immune response have one or two domains
penetrating the cell membrane (transmembrane) (Fig. 2.11). A typical im-
munoglobulin (for example antibody) is composed from four protein chains:
two heavy (H) and two light (L). Variable region in both chains, directed
outside of the cell, is denoted by the letter V (VH and VL domains). The
more constant region is denoted by CH and CL . Variable region is an antigen
binding site. The signal inside the cell is usually transmitted by a protein ty-
rosine kinase (an enzyme transferring a phosphate group on tyrosine), which
is located on the cytoplasmic side of the cell membrane (ch. 8.4.1).

Fig. 2.11 Schematic representation of the structure of the immunoglobulin family

proteins
2.4 Proteins Functions 27

2.4.2.2 Cell Surface Receptors

Many membrane proteins are cell surface receptors. They take part in com-
munication between the cell and the outside environment. Extracellular sig-
naling molecules attach to the receptor, triggering changes in the function
of the cell. This process is called signal transduction (ch. 8). Membrane re-
ceptors are often classified based on their molecular structure or membrane
topology. The polypeptide chains of the simplest are predicted to cross the
lipid bilayer only once, while others cross as many as seven times, for example,
the so-called G protein-coupled receptors (Fig. 2.12). They are a large group
of proteins. They could be activated by agonists or blocked by antagonists.
Photons, fragrances, pheromones, opiates, low molecular weight compounds
such as amine, amino acids, ions, peptides and proteins, lipids and others
could bind to the G protein-coupled receptor (ch. 8.3).

Fig. 2.12 Schematic arrangement of a membrane receptor which associates with

G protein (β-adrenergic receptor)

Membrane receptors include also enzyme-linked receptors (usually single-

pass transmembrane proteins; ch. 8.4) and ion channel linked receptors (mul-
tipass transmembrane proteins; ch. 8.2).

2.4.2.3 Ion Channels

Ion channels are created by a protein or proteins forming pores in the cell
membrane. There are over 300 types of ion channels in a living cell. Their
function is to generate and sustain a small potential gradient between the
inner and outer surface of the cell membrane. Ion channels are present on all
membranes of living organisms. Passage of ions through some of ion channels
is dependent on the ion charge only. Pore diameter in such channels is just one
or two atoms wide at its narrowest point. In some channels, passage through
28 2 Protein Structure and Function

the pore is governed by a gate, which may be opened (activated) or closed

(inactivated) by chemical or electrical signals, temperature, or mechanical
force. Ion channels may be classified by the nature of their gating, the species
of ions passing through those gates or the number of gates (see also ch. 8.2).
Proper activity of ion channels is particularly important in the nervous
system, where they enable the propagation of nerve signals through synapses.
Some predators produce toxins paralyzing the nervous system of their victims
by affecting ion channels (venoms produced by e.g. spiders, scorpions and
snakes). Ion channels are also a key component of the biological processes
that require rapid changes in the cell, such as muscle contraction, transport
of nutrients and ions, the activation of T cells, and insulin secretion from
pancreatic beta cells.

2.4.3 Structural Proteins

Structural proteins are less “active” than those involved in catalyzing reac-
tions, signaling cells, and transporting molecules. They confer stiffness and
rigidity to otherwise-fluid biological components. Most structural proteins
are fibrous proteins. Although actin and tubulin are globular and soluble as
monomers, they polymerize to form long, stiff fibers that make up the cy-
toskeleton. Collagen and elastin are components of connective tissue (e.g.
in the skin), and keratin builds hair, nails, feathers, hooves, and some ani-
mal shells. When the aging process begins the human body stops producing
elastin. Without elastin replenishment, collagen begins to lose its elasticity
and begins to weaken. It results in the loss of the skin’s tone and resiliency.
Thus, some companies advertise the use of collagen and elastin in their anti-
aging creams and moisturizers; however, these products work only on the
skin surface.
Structural proteins are also essential components of muscles (motor pro-
teins such as myosin, kinesin, and dynein as well as actin), and are necessary
to generate the force which allows muscles to contract and move. This class
of proteins is also called contractile proteins.

References
1. Berg, J.M., Tymoczko, J.L., Stryer, L.: Protein Structure and Function. In:
Biochemistry, 5th edn., W.H. Freeman, New York (2002)
2. King, M.W.: Basic Chemistry of Amino Acids. In: themedicalbiochemistry-
page.org, LLC (1996-2012), http://themedicalbiochemistrypage.org/
amino-acids.php
3. King, M.W.: Enzyme Kinetics. In: themedicalbiochemistrypage.org, LLC
(1996-2012), http://themedicalbiochemistrypage.org/
enzyme-kinetics.php
References 29

4. King, M.W.: Protein Structure and Analysis. In: themedicalbiochemistry-

page.org, LLC (1996-2012), http://themedicalbiochemistrypage.org/
protein-structure.php
5. Lodish, H., Berk, A., Zipursky, S.L., et al.: Protein Structure and Function. In:
Molecular Cell Biology, 4th edn. W.H. Freeman, New York (2000)
6. Petsko, G.A., Ringe, D.: Protein Structure and Function. New Science Press
Ltd., London (2004)
7. Vargas-Parada, L. (ed.): Proteins and Gene Expression. In: Miko, I. (ed.) Cell
Biology. Nature Education (2011), http://www.nature.com/scitable/topic/
proteins-and-gene-expression-14122688
Chapter 3
Nucleic Acids

Nucleic acids are polymers formed from nucleotides. Deoxyribonucleotides

build DNA (deoxyribonucleic acid), and ribonucleotides build RNA (ribonu-
cleic acid). With the exception of a few viruses, DNA forms the genetic mate-
rial in all living organisms. Whereas, the major role of RNA is participation
in protein synthesis.

3.1 Nucleotides
Nucleotide consists of three parts: a monosaccharide containing five carbon
atoms (pentose), a nucleobase, and a phosphate group (Fig. 3.1). In nucleotide
present in DNA and RNA there is one phosphate group linked with pentose,
whereas free nucleotides can include two (e.g. ADP — adenosine diphosphate)
or three (e.g. ATP — adenosine triphosphate) phosphate groups. Nucleotide,

Fig. 3.1 The basic structure of the nucleotide

W. Widłak: Molecular Biology, LNBI 8248, pp. 31–48, 2013.


c Springer-Verlag Berlin Heidelberg 2013
32 3 Nucleic Acids

in which all phosphate groups are removed (i.e. only the nucleobase and
pentose) is called a nucleoside.

3.1.1 Pentoses
Pentoses are monosaccharides with five carbon atoms. Saccharides (carbo-
hydrates) are built of carbon chains with side hydroxyl groups (-OH) and a
carbonyl group (>C=O) of aldehyde type (aldose) or ketone type (ketose).
Ribose is an aldopentose, which is present in nucleic acids in the form of a
ring (Fig. 3.2). Ribose is a component of RNA. The DNA contains deoxyri-
bose, which means that in DNA ribose is lacking an oxygen atom at carbon 2
(2’-deoxyribose). Because of that RNA is called “ribonucleic acid” and DNA
— “deoxyribonucleic acid”.

Fig. 3.2 The chemical structure of pentose ring forms: five carbon atoms are la-
beled from the C1’ to C5’. In RNA ribose is present, in DNA — deoxyribose,
which lacks an oxygen atom at C2’.

3.1.2 Nitrogenous Bases of Nucleotides

Nucleotides present in nucleic acids are built of five different nucleobases
denoted with the single letter code. They are: Adenine (A), Cytosine (C),
Guanine (G), Thymine (T) and Uracil (U).
A, C, G and T are present in DNA; A, C, G and U are present in RNA.
A and G are built of two rings joined together — they are called purines.
C, T and U contain only one ring — they are called pyrimidines (Fig. 3.3
and 3.4).
Nucleobases are connected with the saccharides by a N-glycosidic bond fo-
rmed between the nitrogen in the purine ring (N-9), or pyrimidine ring (N-1)
and the hydroxyl group at carbon 1 (C-1’) in pentose. Table 3.1 gives the
names of existing bases and their derivatives.
3.1 Nucleotides 33

(a) (b)

Fig. 3.3 General formulas of purine (a) and pyrimidine (b)

Fig. 3.4 The chemical structure of nucleobases found in DNA and RNA. Green
pentagon shows the location of pentose.

Table 3.1 The names of the bases, nucleosides, and nucleotides

 

 
 
  
 
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34 3 Nucleic Acids

3.2 DNA Properties

3.2.1 Nucleotide Chain
In the nucleotide chain, nucleotides are connected with each other by a
phosphodiester bond, which is formed by the condensation reaction cat-
alyzed by polymerases or ligases (Fig. 3.5). Ligation, i.e. connection of two
chains of nucleic acids is catalyzed by ligases, while the synthesis of a nu-
cleic acid chain (by attaching nucleotides) is catalyzed by RNA polymerase
or DNA polymerase. The phosphate group, forming phosphodiester bonds in
the chain of nucleic acids, binds with hydroxyl groups at carbons 3’ and 5’
of adjacent pentose rings (there are also possible other types of phosphodi-
ester bonds in nucleotides, such as an intramolecular bond in so-called cyclic
monophosphate; Fig. 8.2).

Fig. 3.5 Formation of a phosphodiester bond by condensation reaction

Like the peptide chain, a nucleic acid chain also has the orientation result-
ing from asymmetric character of bonds. 5’ end has a free phosphate group
3.2 DNA Properties 35

at carbon C5’ of pentose, 3’ end has a free hydroxyl group at carbon C3’
of pentose (Fig. 3.6). Synthesis of the nucleic acid chain always proceeds in
the direction from 5’ to 3’. Because of that, unless it is stated otherwise, the
sequence of nucleotides in the nucleic acid chain is written from the 5’ to
3’ end (from left to right). The sequence of nucleobases along the backbone
encodes information. This information is read using the genetic code (ch. 3.5),
which specifies the sequence of the amino acids within proteins. The code is
read by copying stretches of DNA into the related nucleic acid RNA in a
process called transcription (ch. 5).

Fig. 3.6 The nucleic acid chain. 5’ end has a free phosphate group (at C5’), the
3’ end — a free hydroxyl group (at C3’).

The DNA molecule is usually built from two chains, while most molecules
of RNA - from a single chain. Length of chain is defined by the number
of bases (or nucleotides) in the chain. In case of double-stranded nucleic
acid nucleobases from neighboring chains create pairs. In this case, the chain
length is given as the number of base pairs (bp). Short chains of nucleic
36 3 Nucleic Acids

acids (less than 50 nucleotides) are defined as oligonucleotides. Longer chains

are polynucleotides.

3.2.2 DNA Structure

The two chains of the DNA molecule are held together by hydrogen bonds
between the “paired” nitrogen bases. Adenine forms two hydrogen bonds with
thymine and cytosine forms three bonds with guanine (Fig. 3.7). Although
other pairs (such as G:T or C:T) also may form hydrogen bonds, they are we-
aker than those in pairs present in DNA (C:G and A:T). Thanks to the spe-
cific base pairing, the DNA strands are complementary with each other. Thus,
the sequence of nucleotides in one strand determines the sequence of comple-
mentary strand. That is why in databases only one strand sequences (in the
orientation 5’ to 3’) are given.

Fig. 3.7 Schematic base-pairing in DNA. Adenine (A) always forms a pair with
thymine (T) and guanine (G) with cytosine (C)

Breaking of hydrogen bonds between the complementary bases leads to

separation of two DNA chains (denaturation, melting). Such effect can be
achieved for example by heating of DNA solution. The temperature at which
loss of half of the double-stranded structure occurs is called the melting tem-
perature (Tm ). When the temperature is lowered, the DNA strands are again
reconnected complementarily. This DNA property is used in hybridization
techniques.
Two polynucleotide chains in a DNA molecule run in opposite directions to
each other (i.e. are arranged anti-parallel). They are spinned around the com-
mon axis forming double-stranded helix. Purine and pyrimidine bases are lo-
cated inside the helix (forming hydrophobic core), while the phosphate groups
3.3 Structure and Function of RNA 37

and deoxyriboses — outside (forming hydrophilic, polar, external backbone).

Double helix, first described in 1953 by James Watson and Francis Crick, is
called B form of DNA. In that form helix is right-handed, a full turn of the
helix takes 3.4 nm and the distance between two neighboring pairs of bases
is 0.34 nm. Intertwined strands create two grooves of different width, called
the major and minor groove (Fig. 3.8). Such a structure facilitates binding
of specific proteins to DNA.

Fig. 3.8 The structures adopted by the DNA double helix. Sourced from:
http://en.wikipedia.org/wiki/DNA

In high salt concentration solutions or after the addition of alcohol, the

structure of DNA is converted to A form. It is also right-handed, but full
turn takes place every 2.3 nm, so one turnover occurs every 11 base pairs.
The A form in the cell may be produced in hybrid pairings of DNA and RNA
strands, as well as in enzyme-DNA complexes. Segments of DNA where the
bases have been chemically modified by methylation may undergo a larger
change in conformation and adopt the Z form, which phosphate backbone
resembles a zigzag. It is a left-handed form of DNA: one turnover takes 4.6 nm
and occurs every 12 base pairs. Such structure of DNA can be recognized by
specific Z-DNA binding proteins and may be involved in the regulation of
transcription (Fig. 3.8).

3.3 Structure and Function of RNA

RNA is synthesized in the cell in the process of transcription on the basis of
information contained in the sequence of DNA. Most RNA molecules retain
a single-stranded form and have a much shorter chain of nucleotides than the
DNA molecule. However, RNA can also form secondary structures such as
38 3 Nucleic Acids

hairpin structure or stem-loop structure (Fig. 3.9). Main classes of RNA are
mRNA (messenger RNA), tRNA (transfer RNA) and rRNA (ribosomal
RNA). The main function of these classes of RNA is participation in pro-
tein synthesis. Moreover, in the cell there are present so called non-coding
RNA (ncRNA), which is not involved in the translation process in a classical
way. What is interesting, a number of RNA molecules shows, like proteins,
catalytic activities. Such enzymatic RNA molecules are called ribozymes.

(a) (b)
Fig. 3.9 Examples of secondary structures of RNA: (a) hairpin structure; (b)
stem-loop structure

3.3.1 Messenger RNA

Messenger RNA (mRNA) carries coding information (copied from DNA
sequence), which is used later for protein synthesis (translation) on ribo-
somes (ch. 6.1). The coding sequence of the mRNA determines the amino
acid sequence in the protein that is produced. Three consecutive nucleotides
(codon) in the mRNA encode an amino acid or form a stop signal for protein
synthesis (Fig. 3.10).

Fig. 3.10 The order of events in the synthesis of proteins: on the DNA tem-
plate mRNA is synthesized in the process of transcription, on the mRNA template
proteins are further produced in the translation process. mRNA sequence is com-
plementary to DNA’s template strand and identical to the coding strand (except
that in RNA T is replaced by U). Commonly, in the case of DNA sequence only
the sequence of the coding strand is given.
3.3 Structure and Function of RNA 39

3.3.2 Transfer RNA

Transfer RNA (tRNA) consists of 74 - 95 nucleotides. It takes a complicated
secondary structure similar to the cloverleaf, where approximately half of the
nucleotides are paired (Fig. 3.11). Some of nucleotides in the tRNA molecule
are methylated or modified in the other way, for example inosine (I), pseu-
douridine (ψ) or dihydrouridine (D). The main task of tRNA is translation
of a sequence of nucleotides in mRNA into the sequence of amino acids in
the peptide. This is possible because in the middle loop of tRNA the anti-
codon is located, which is complementary to the codon present on mRNA.
3’ end of tRNA carries amino acid appropriate to ascribed codon (specific
aminoacyl-tRNA is formed) (ch. 6.1.2).

Fig. 3.11 Secondary structure of tRNA. Unusual or modified nucleotides are

marked in blue (m - methylated). Anticodon is a triplet of nucleotides comple-
mentary to codon in the mRNA. When amino acid binds to the 3’ end of tRNA,
the formed molecule is called aminoacyl-tRNA.

3.3.3 Ribosomes and rRNA

In prokaryotic cells ribosomal RNA (rRNA) of three types (23S, 5S, and
16S) are present. In mammalian cells there are four types of rRNA: 28S,
5.8S, 5S, and 18S (unit “S” stands for Svedberg and it represents measure
40 3 Nucleic Acids

of sedimentation rate). rRNA is produced in the nucleus and transported

to the cytoplasm, where it creates complexes with specific proteins, forming
ribosome. Ribosome in 2/3 is composed of RNA and 1/3 of protein. It has
two subunits: large and small. During protein synthesis the ribosome binds
mRNA and tRNA, as shown in Fig. 3.12. Only tRNA which contains the
anticodon matching to mRNA codon (located centrally between units of the
ribosome) may connect to the complex.

Fig. 3.12 The mRNA – ribosome – aminoacyl-tRNA complex formed during

synthesis of protein

3.3.4 Non Coding RNA

In addition to the mentioned above classes of RNA participating directly in
the process of protein synthesis, a range of other classes of RNA are produced
in cells. They are called small non-coding RNA molecules. These include:

– snRNA (small nuclear RNA); low molecular weight nuclear RNA with a
length up to 300 nt — involved in mRNA processing;
– snoRNA (small nucleolar RNA); low-molecular nucleolar RNA — respon-
sible for the modification of ribosomal RNA;
– miRNA (micro RNA) and siRNA (small/short interfering RNA) — in-
volved in gene expression regulation.
In recent years, the special interest is above siRNA molecules, due to the fact
that they are routinely used in laboratories around the world to silence gene
activity at the mRNA level. Both miRNA and siRNA have a length of 18
to 25 nucleotides, and their effect in the cell is similar (Fig. 3.13). There is
consensus in the literature for the definition of miRNA and siRNA. Generally
name miRNA is reserved for the molecules produced endogenously (i.e. inside
the cell), siRNA — for delivered to the cell from outside. miRNA is formed
from larger precursor (pri-miRNA), transcribed in the nucleus by polymerase
3.3 Structure and Function of RNA 41

RNA II (which is also involved in transcription of mRNA). Such transcript is

trimmed and transported to the cytoplasm, where it arrives in the form of ap-
proximately 70-nucleotide pre-miRNA forming stem-loop structure. The loop
is removed leaving miRNA duplex. Base pairing in miRNA duplex is not per-
fect, so miRNA does not have 100% sequence homology to the target mRNA.
siRNA is formed either from dsRNA (double standed RNA) or from shRNA
(small/short harpin RNA) coming from viruses, transposons or delivered to
cells experimentally. Like in the case of miRNA, initially siRNA duplex is
formed. It has 100% homology to sequence of the target mRNA. miRNA or
siRNA duplexes are then separated to single strands. One strand is removed,
the other inhibits the translation of complementary mRNA (miRNA) or initi-
ates its degradation (siRNA). As a result expression of the gene is diminished
(encoded protein is not created). Inhibition of expression associated with the
presence of miRNA is likely to occur also through the induction of epige-
netic gene silencing (by methylation of histones, which leads to chromatin
condensation).

Fig. 3.13 miRNA and siRNA metabolism in the cell. Altered from:
http://www.nature.com/cr/journal/v15/n11/fig_tab/7290371f1.html
42 3 Nucleic Acids

The phenomenon of silencing or exclusion of gene expression by double-

stranded RNA with a sequence similar to the target DNA sequence is called
RNA interference (RNAi). It has an important role in defending cells aga-
inst parasitic genes — viruses and transposons. On the other hand, miRNA
are involved in negative regulation of gene expression during development.
miRNA are involved in regulation of about 30% of human genes.
For the discovery of RNA interference in 1998, american researchers An-
drew Z. Fire and Craig C. Mello received in 2006 Nobel Prize in Physiology
or Medicine.

3.4 The Organization of Genetic Material in the

Nucleus - Chromatin
Genomic DNA in the eukaryotic nucleus is hierarchically packaged into chro-
matin. Chromatin is a fibrous substance, which at the time of cell division
becomes visible in the form of chromosomes (ch. 1.3.1). It is composed of
DNA, RNA, histones, and other proteins. Each human cell has 1.8 meters of
DNA. The role of chromatin is to package DNA into a smaller volume to fit
in the nucleus and to control gene expression and DNA replication. Processes
like DNA repair, replication, recombination, and transcription take place
in the chromatin. Chromatin structure is changed dynamically: in regions
where transcription takes place it is less condensed, loose (euchromatin), in
transcriptionally inactive regions it is more condensed (heterochromatin)
(Fig. 3.16).

3.4.1 Histones and Nucleosomes

The basic structural element of chromatin is nucleosome, which consists
of 146 bp DNA and eight histone molecules (so-called histone octamer;
Fig. 3.14). Histones are highly conserved alkaline proteins closely associated
with DNA. They are responsible for the structure of chromatin and are in-
volved in the regulation of gene expression. Nucleosome consists of 2 copies of
each of histone proteins H2A, H2B, H3, and H4. They form the core, around
which the DNA is wrapped forming two loops. The fifth type of histone,
histone H1, which is more loosely connected with DNA, binds nucleosomes
and is involved in formation of more complex chromatin structures. Inter-
actions between histones and DNA occur between positively charged lysine
side chains (present in large amounts in the N-terminus of histone) and neg-
atively charged phosphate groups in DNA. If lysine is acetylated, its charge
is neutralized. This leads to weakening of histones binding with DNA and
plays a crucial role in the regulation of transcription (ch. 5).
3.4 The Organization of Genetic Material in the Nucleus - Chromatin 43

Fig. 3.14 Nucleosome structure

3.4.2 Chromatin Structure

In condensed chromatin each nucleosome binds with the next one by linker
histone H1. This leads to the creation of 30 nm chromatin fiber (solenoid)
(Fig. 3.15). In transcriptionally active chromatin, histone H1 is released from
DNA and nucleosomes can change position, which means they move along
the DNA strand. Nucleosome which never changes the position is called po-
sitioned nucleosome.

(a) (b)

Fig. 3.15 Stages of chromatin organization: (a) nucleosomes fiber (“beads-on-a-

string”); (b) single turn of solenoid (30 nm fiber)

Solenoid forms further more complex structures ensuring multilevel or-

ganization of chromatin in the nucleus. Important level of organization of
chromatin are so called domains or loops, which are formed by interactions
between nucleosome fibers and protein structures in nucleus (nuclear skele-
ton). The highest level of compaction of chromatin are chromosomes condens-
ing during cell division (Fig. 3.16). The compacted DNA molecule is 40,000
times shorter than an unpacked.
44 3 Nucleic Acids

Fig. 3.16 Model of chromatin compaction and accompanying changes in gene

activity. Altered from. http://en.wikipedia.org/wiki/
File:Chromatin_Structures.png

Recently is has been shown that 30 nm fibers are not detected in chro-
mosomes in situ (i.e. examined exactly in the cell). It was hypothesised that
crowding by cytoplasmic macromolecules is a sufficient factor that enables
the packing of metaphase chromosomes.

3.5 Genetic Code

The genetic code is a set of rules, by which genetic information encoded in the
nucleotide sequence of DNA or RNA may undergo translation into proteins
(amino acid sequences) by living cells (Fig. 3.17). Translation takes place
based on information stored in mRNA, which is built from nucleotides, while
proteins are composed of amino acids. That is why it is necessary to link the
exact nucleotide sequence with the amino acid sequence. The relationship
between these sequences was deciphered in the 1960s by Marshall Nirenberg
and his team (Nobel Prize in Physiology or Medicine in 1968). They correctly
assumed that three successive nucleotides (codon) code for one amino acid.
The most important characteristic of the genetic code is its unambiguity,
which means that a particular codon may encode only one type of amino
acid. Genetic code is not strictly defined — an amino acid may be encoded
by several different codons (we say that the genetic code is degenerated). If
amino acid is encoded by several codons, the first two positions are usually
the same, and differences exist at the third position. Moreover, amino acids
with similar physical characteristics have similar codons. That is why point
mutations (ch. 4.4) in DNA at the third position of codon usually does not
change the sequence of encoded protein. Mutations at the first or second
position more frequently change the amino acid, but more than likely it will
have similar properties.
3.6 Genes and Genome 45

Fig. 3.17 The standard genetic code (codons should be read starting from the
central ring). A peptide synthesis always begins with methionine (Met) encoded
by the codon AUG, which encodes also methionine located inside the peptide
chain. Stop codons (UAA, UAG, UGA) terminate peptide synthesis. In the diagram,
nucleotides typical for RNA are given (in DNA U should be replaced by T). The
DNA sequence from a start codon (ATG) to a stop codon (TAA, TAG or TGA)
is called an open reading frame (ORF), which encodes a protein. Amino acids
marked by ∗ can be encoded by several codons varying at the first nucleotide (for
example arginine, Arg, can be encoded by AGA, or AGG as well as by CGA, CGG,
CGU or CGC; different codons for the same amino acid can even appear side by
side in the same molecule of mRNA).

Genetic code is universal for almost all organisms. There are however sev-
eral exceptions to the rules of the standard genetic code. For example in
the mitochondrial DNA of many organisms or in the nuclear DNA of several
lower organisms there are different start or stop codons.

3.6 Genes and Genome

The definition of a gene has changed over time, starting with the definition
introduced by Gregor Mendel in the nineteenth century. One of the most
popular, although not the actual definition, was that a gene is a fragment of
DNA encoding a protein chain. Currently it is assumed that the gene is an
entire sequence of the DNA necessary to built a protein or RNA. The sequence
of the gene can be divided into the regulatory region and the transcribed
46 3 Nucleic Acids

region. The regulatory region can be distant or can be located directly next
to the region of transcription. In eukaryotic organisms the transcribed region
usually contains exons and introns (in prokaryotic organisms genes do not
have introns). Exons encode functional proteins or RNA, while introns are
removed after transcription (ch. 5). In DNA, in addition to genes, pseudogenes
are present. They are non-functional genes. Often they are formed as a result
of duplications and mutations of normal genes. Regions coding for functional
proteins or RNA represent only about 3% of the human genome.
All genetic information of an organism is called its genome. For most
organisms, it is a complete DNA sequence (Table 3.2). Exceptions are RNA
viruses, in which a complete RNA sequence is their genome. With the term
of genome, the term of proteome is associated, which comprises all proteins
present in a cell at a given time. All body cells have the same genome, but can
differ significantly with proteome. The proteome is larger than the genome,
as there are many more proteins than genes. This is due to an alternative
splicing of genes and post-translational modifications (ch. 5.8).

3.7 Repetitive Sequences

DNA sequences containing most genetic information in the form of structural
genes are described as unique DNA. They are found in the genome in two
copies - alleles located in corresponding parts of homologous chromosomes.
However, many DNA sequences repeat several times in the total cellular
DNA. For example, in the telomeres located at the ends of chromosomes, the
sequence “TTAGGG” repeats thousands of times throughout about 15,000 bp.
Such sequences are called repetitive sequences. The function of repetitive

Table 3.2 Variation in genome size and gene content

Organizm Genome Number

type Organism size (Mb*) of genes
Viruses Hepatitis D 0.0017 2
Hepatitis B 0.0032 4
HIV-1 0.0092 9
Bacteriophage λ 0.0485 90
Bacterium Escherichia coli 4.6 4,437
Yeast Saccharomyces cerevisiae 12.1 6,300
Nematode Caenorhabditi elegans 100 19,000
Insect Drosophila melanogaster (fruit fly) 130 14,000
Mammals Mus musculus (house mouse) 3,000 20,000-30,000
Homo sapiens (human) 3,200 20,000-30,000
∗
1 Mb = 1 million base pairs (in the case of double-stranded DNA or RNA) or 1
million bases (in the case of single-stranded DNA or RNA).
References 47

DNA is largely unknown. The repetitive DNA may be clustered at one place
or interspersed with unique sequences, each repeat unit ranging from a few
hundred to few thousand base pairs.
Classification of repetitive sequences was originally based on kinetics of
reassociation of denatured DNA. The total DNA is firstly randomly digested
into fragments of average size of about 1,000 bp. Then the DNA fragments in
aqueous solution are heated and complementary strands dissociate to single
strands (due to breaking of hydrogen bonds). Decreasing of the temperature is
connected with reassociation of DNA sequences (hybridization) according to
the rule of complementarity of bases. Reassociation kinetics can be estimated
for example by measuring of changes in absorbance of the solution at 260 nm.
The absorption coefficient of the double-stranded DNA is about 40% lower
than of single-stranded DNA. If DNA fragment contains a sequence that re-
peats several times in the genome, it has a greater chance to find a matching
strand and reassociate faster than other fragments containing less repetitive
sequences. Relying on the rate of reassociation, the DNA sequences were
divided into three classes:
1. Highly repetitive: hybridizing very quickly (just a few seconds). In mam-
mals they make up 10-15% of the DNA. Short sequences called microsatel-
lites and tandem repeats are classified here.
2. Moderately repetitive: hybridizing in the intermediate speed. Represent
25-40% of the genome of mammals. Among these are interspersed repeats,
also known as mobile elements or transposons.
3. Single or of small number of repetitions: represent 50-60% of mammalian
DNA.
Interspersed repeats can be a short sequences of length 100-500 bp, called
SINES (Short Interspersed Nuclear Elements) or long repetitions, with a
length of several thousand base pairs, called LINES (Long Interspersed Nu-
clear Elements). An example of a sequence belonging to SINES are highly
homologous Alu sequences with a length of 300 bp. In human genome they are
present at level close to a million copies. They are dispersed throughout the
genome and can serve as initiation sites of DNA replication. Another class of
repetitive DNA often occurs in the centromeres (satellite DNA). In Drosophila
satellite DNA is composed of seven-nucleotide sequence repeated more than
10,000 times. The genes encoding the 5.8S, 18S, and 28S rRNA appear in
groups located in the nucleolus, and are repeated tandemly many times. The
genes encoding histones are also found in tandemly repeated groups.

References
1. Berg, J.M., Tymoczko, J.L., Stryer, L.: DNA, RNA, and the Flow of Genetic
Information. In: Biochemistry, 5th edn. W.H. Freeman, New York (2002)
2. Clancy, S.: Chemical structure of RNA. Nature Education 1(1) (2008), http://
www.nature.com/scitable/topicpage/chemical-structure-of-rna-348
48 3 Nucleic Acids

3. Clancy, S.: RNA functions. Nature Education 1(1) (2008),

http://www.nature.com/scitable/topicpage/rna-functions-352
4. Hancock, R.: Structure of metaphase chromosomes: a role for effects of macro-
molecular crowding. PLoS One 7(4), e36045 (2012)
5. King, M.W.: Basic Chemistry of Nucleic Acids. In: themedicalbiochemistry-
page.org, LLC (1996-2012), http://themedicalbiochemistrypage.org/
nucleic-acids.php
6. Maeshima, K., Hihara, S., Eltsov, M.: Chromatin structure: does the 30-nm
fibre exist in vivo? Curr. Opin. Cell Biol. 22, 291–297 (2010)
7. Lodish, H., Berk, A., Zipursky, S.L., et al.: Nucleic Acids, the Genetic Code,
and the Synthesis of Macromolecules. In: Molecular Cell Biology, 4th edn. W.H.
Freeman, New York (2000)
8. Phillips, T.: Small non-coding RNA and gene expression. Nature Education 1(1)
(2008), http://www.nature.com/scitable/topicpage/
small-non-coding-rna-and-gene-expression-1078
9. Pray, L.: Eukaryotic genome complexity. Nature Education 1(1) (2008),
http://www.nature.com/scitable/topicpage/
eukaryotic-genome-complexity-437
10. Yeung, M.L., Bennasser, Y., Le, S.Y., et al.: siRNA, miRNA and HIV: promises
and challenges. Cell Research 15, 935–946 (2005)
Chapter 4
DNA Replication, Mutations,
and Repair

4.1 DNA Replication Mechanism

Replication (i.e. copying) of DNA takes place before cell division. During
replication one double-stranded DNA molecule produces two identical copies.
Each strand of the original double-stranded DNA molecule serves as a tem-
plate for the production of the complementary strand, a process referred to as
semiconservative replication. Replication is triggered by a transcription fac-
tor, in yeast called MCB binding factor, in mammals — E2F, that regulates
the expression of enzymes necessary for replication: DNA polymerases, DNA
primases and cyclins. Replication starts in a specific place (called replica-
tion origin) (Fig. 4.1). In genomic DNA of E. coli there is only one place of
replication origin (called oriC ), whereas eukaryotic DNA has many such sites
on each chromosome. In a sequence of replication origin, A:T pairs dominate,
which are easier to separate than a C:G pairs, because they are connected
only by two hydrogen bonds. Unwinding of DNA at the origin and synthesis
of new strands leads to the formation of a replication fork (Fig. 4.2).

Fig. 4.1 Sequences of bacterial and yeast replication origin: (a) in bacteria it is
a 245 bp sequence containing three 13 bp tandemly repeated segments of almost
identical sequence (green), and four 9 bp segments with high sequence similarity
(yellow); (b) yeast replication origin sequence is called ARS (autonomously repli-
cating sequence). The A region is essential for the initiation of replication, while
B1, B2 and B3 enhance efficiency of replication.

W. Widłak: Molecular Biology, LNBI 8248, pp. 49–69, 2013.


c Springer-Verlag Berlin Heidelberg 2013
50 4 DNA Replication, Mutations, and Repair

Fig. 4.2 Scheme of DNA replication in eukaryotes. O1, O2 and O3 — replication

origins for replicons R1, R2 and R3. Replication requires unwinding of double he-
lix. New strands are synthesized by DNA polymerase on template of old strands.
Unwinded DNA forms replication fork, moving in one direction, thus “replication
bubble” increases. Replication begins at some replication origins earlier than at
others. As replication nears completion, “bubbles” of newly replicated DNA meet
and fuse, finally forming two new molecules.

Replication occurs on both DNA strands simultaneously. E. coli needs 42 mi-

nutes to replicate the whole genomic DNA. The speed of the replication fork
progression, formed by unwinded DNA, is about 1,000 bp per second. In euka-
ryotes, it moves much slower — only 50-100 bp per second. This is due to the
association of DNA with histones. Replication of the entire human genome
takes approximately 8 hours, Drosophila genome — only 3-4 minutes (thanks
to the 6,000 replication forks operating simultaneously on a DNA molecule).
A DNA molecule is synthesized by DNA polymerases from deoxyribonucle-
oside triphosphates (dNTPs). The process is similar to the synthesis of RNA
(ch. 5). Both polymerases, DNA and RNA, synthesize the nucleic acid chain
only in the 5’ to 3’ direction. Because two chains of DNA are anti-parallel,
only one strand (leading strand) can be synthesized by DNA polymerase co-
ntinuously. The second strand (lagging strand) is synthesized in fragments
(Fig. 4.3). Cellular proofreading and error-checking mechanisms ensure near
perfect fidelity for DNA replication.

4.1.1 DNA Polymerases

DNA polymerases are enzymes that carry out all forms of DNA replication.
DNA polymerase synthesizes a new strand of DNA by extending the 3’ end
of an existing nucleotide chain, adding new nucleotides complementary to the
4.1 DNA Replication Mechanism 51

Fig. 4.3 Synthesis of lagging strand: (a) comparison of the leading strand and
lagging strand; (b) primase synthesizes a new primer of a length of about 10 nt.
The distance between two primers is about 1,000-2,000 nt in bacteria, and about
100-200 nt in eukaryotic cells; (c) DNA polymerase attaches nucleotides to the new
primer in the direction 5’ to 3’ until it encounters the 5’ end of the previous primer.
Newly synthesized DNA fragment is called Okazaki fragment (named after their
discoverer); (d) in E. coli DNA polymerase I shows exonuclease activity and from
the 5’ end removes encountered primer and fills created gap; (e) DNA ligase joins
adjacent Okazaki fragments. The entire lagging strand is synthesized by repeating
steps (b) to (e).

template strand. In E. coli there are three types of DNA polymerases: I, II and
III. DNA polymerase I completes gaps between the fragments of DNA formed
during the synthesis of the lagging strand. It is also involved in DNA repair.
DNA polymerase II is activated in response to DNA damage (SOS response).
The majority of DNA in prokaryotic cells is synthesized by DNA polymerase
III. This enzyme is 900 kDa in size, and is composed of several subunits (they
form so called holoenzyme). The polymerase core is made up of α, ε, and θ
subunits. The role of other units is to prevent detachment of the enzyme
from the template. Two β units (β2 ) act as sliding DNA clamps, keeping the
polymerase bound to the DNA. Together with the polymerase core they slide
along the DNA molecule. It allows a continuous synthesis of approximately
5 × 105 nucleotides. In the absence of β subunits the polymerase core is
52 4 DNA Replication, Mutations, and Repair

detached after synthesis of about 10-50 nucleotides. DNA polymerase III

can add about 1,000 nucleotides per second and it is extremely precise: ε
subunit, which is 3 → 5 exonuclease, checks continuously the correctness of
the replication.
In mammals there are five main DNA polymerases (α, β, γ, δ and ε) and
several smaller. γ polymerase replicates mitochondrial DNA, the others are
present in the nucleus, where they perform different functions:
– α: lagging strand synthesis;
– β and ε: DNA repair;
– δ: leading strand synthesis.
To start the synthesis of DNA a short RNA molecule (primer) is required.
The primer is about 5-12 nucleotides in length, and is complementary to one
strand of the template. It is synthesized by a specific enzyme — primase,
which is the RNA polymerase. The use of ribonucleotides for the synthesis
of primer means that this fragment is temporary — it is removed in the
final phase of replication. RNA polymerases, unlike DNA polymerases, can
synthesize new strand without a primer, because they do not check whether
the nucleotide inserted into the chain is correct (they are much less accurate).
Other proteins involved in replication are helicases and single-strand DNA
binding (SSB) proteins. Helicase unwinds double helix and SSB proteins bind
to single-stranded fragments, preventing from the duplex restoration.
The mechanism of replication in bacteria and eukaryotes is similar. How-
ever, eukaryotic DNA polymerases do not possess subunits similar for bac-
terial β subunit. When they attach to DNA, they use the PCNA protein
(proliferating cell nuclear antigen).

4.1.2 Polymerase Chain Reaction (PCR)

The ability of DNA polymerase to produce identical copies of DNA is utilized
in the laboratory to amplify fragments of DNA (the target sequence is used
many times as a template). Such strategy, named polymerase chain reaction
(PCR), is the most sensitive method to detect and amplify a target sequence.
Nowadays PCR is widely used for many applications, independently or in
combination with other techniques.
The polymerase can synthesize a new strand only on a single stranded tem-
plate, thus the process takes place after DNA denaturation (see ch. 3.2.2).
In vitro, in a tube, it can be achieved by heating the DNA solution. Poly-
merases of most organisms cannot survive in high temperatures, however in
PCR, DNA polymerase from organisms living in hot springs is used, such as
Taq polymerase from Thermus aquaticus. To start the synthesis, the DNA
polymerase needs a primer. In PCR, two primers complementary to the tar-
get sequence are used (each complementary to a different strand). The se-
quence flanked by primers is amplified after consecutive PCR cycles (Fig. 4.4).
4.2 Telomerases and Cellular Senescence 53

Fig. 4.4 Scheme of polymerase chain reaction

Products generated in one cycle are used as additional templates in the next
cycle. Up to 40 cycles (consisting of three steps: denaturation, primers an-
nealing and chain elongation) could be processed in PCR machine. After 30
cycles more than 1 × 109 molecules are already produced from one DNA
molecule.

4.2 Telomerases and Cellular Senescence

Synthesis of lagging strand requires synthesis of multiple primers, that are
finally removed. In bacteria, which have a circular DNA molecule, there is no
problem with the replication of the whole molecule. In case of linear DNA
54 4 DNA Replication, Mutations, and Repair

molecule, lagging strand is always shorter than the template (at least by the
length of the primer). Such situation takes place at the end of chromosomes.
At the ends of eukaryotic chromosomes telomere regions are found, which
minimize the problem of shortening of the DNA strands during every cell di-
vision (the second function of telomeres is to protect chromosomes from fusing
with each other). Telomeres contain hundreds of tandem repeated sequences
(ch. 3.7), thus their shortening after each cell division is not detrimental to
the cell. Cells can divide a certain number of times before the DNA loss pre-
vents further division (this is known as the Hayflick limit). However, after
reaching a certain telemere length, the cell may stop to divide and die. This
is a normal process in somatic (body) cells. Steady shortening of telomeres
with each replication in somatic cells may have a role in senescence and in
the prevention of cancer.
Within the germ cell line, which passes DNA to the next generation, the
repetitive sequences of the telomere region are extended by telomerase. It is
a large ribonucleoprotein enzyme that catalyzes the elongation of 3’ end of
DNA (Fig. 4.5). Telomerase contains internal RNA molecule, which serves as
a template (Fig. 4.6). So telomerase is a reverse transcriptase with its own
template. That is quite unique: any other of the known polymerases does not
contain polynucleotide template. Telomerase can become mistakenly active
in somatic cells, sometimes leading to cancer formation.

Fig. 4.5 Scheme of telomere elongation by telomerase. Telomerase first extends

the longer strand of DNA. Then, using the same mechanism as synthesizing the
lagging strand, the shorter strand is extended.

4.3 Topoisomerases
During DNA replication, DNA ahead of the replication bubble becomes posi-
tively supercoiled, while DNA behind the replication fork becomes entangled
forming precatenanes (Fig. 4.7). Enzymes called topoisomerases play an es-
sential role in resolving these topological problems.
4.3 Topoisomerases 55

Fig. 4.6 The mechanism of telomere extension by telomerase. In human chromo-

some telomere has about 10-15 thousand base pairs, which consist of many tandemly
repeated sequences GGGTTA. Telomerase contains RNA complementary to repeti-
tive sequences in the telomere, which is used as a template for the synthesis of DNA.
By repeating a cycle of DNA synthesis and telomerase translocation, telomere is
extended by many repeats.

(a) (b) (c) (d)

Fig. 4.7 The structure of catenane and supercoils of DNA: (a) circular cate-
nane; (b) positive supercoil DNA (left-handed); (c) negative supercoil DNA (right-
handed); (d) positive supercoil bacterial DNA during replication. Supercoil DNA
molecule is more dense than the relaxed DNA of the same length.
56 4 DNA Replication, Mutations, and Repair

There are two types of topoisomerases: type I creates a transient break in

one strand of DNA, type II — cuts both strands of one DNA double helix,
passes another unbroken DNA helix through it, and then reanneals the cut
strand (Fig. 4.8). Type II is more efficient in removing supercoils, however it
requires energy from ATP hydrolysis. Type I does not require such energy.
Topo I in prokaryotes and eukaryotes is a type I topoisomerase. Examples of
type II topoisomerases include eukaryotic topo II, E. coli gyrase and topo IV.

(a) (b)

Fig. 4.8 Topo II functions: (a) removing of supercoils; (b) separation of linked
molecules of catenanes. These actions require cutting of both strands of DNA
(marked with black line). After relocation of the DNA strands they are again con-
nected.

Eukaryotic topo I and topo II can relax both positively and negatively su-
percoiled DNA. In bacteria topo I catalyzes only the relaxation of negatively
supercoiled DNA. Bacterial gyrase has two functions: relaxes positively super-
coiled DNA during replication and forms a negative twists in the DNA (so
it can be packed into a cell). During replication, these negative turns are
removed by topo I.
Without topoisomerases DNA cannot replicate correctly. That is why
inhibitors of topoisomerases are used as anticancer drugs that inhibit cell
division. Such drugs inhibit the division of all cells in the body, including
normal cells. Thus, for example hair loss is a common side effect of therapy
(since hair are growing due to a permanent division of cells in hair follicles).
DNA-damaging effect of topoisomerases inhibitors, outside of its potentially
curative properties, may lead to secondary neoplasms in the patient.

4.4 Mutations and Their Consequences

A mutation is a permanent change in the DNA sequence concerning several
nucleobases or changes on the scale of the chromosome. Effects of the mu-
tation could be observed only when it occurs in gene exons or regulatory
elements (ch. 5.3). Changes in the non-coding regions of DNA generally do
not affect function. If a mutation occurs in a germ-line cell (i.e., egg or sperm
cells), it can be passed to an organism’s offspring. In contrast to germ-line
mutations, somatic mutations occur in cells found elsewhere in an organism’s
4.4 Mutations and Their Consequences 57

body. Such mutations are passed to daughter cells, but they are not passed
to offspring.
In dipliod organisms, which have two copies of each gene (alleles), mu-
tations are recessive or dominant. Recessive mutations inactivate the af-
fected gene and lead to a loss of function. Thus, to give rise to a mutant
phenotype, both alleles must carry the mutation (otherwise the correct allele
will provide a proper function). In contrast, the phenotypic consequences of
a dominant mutation are observed in a heterozygous individual carrying one
mutant and one normal allele. Dominant mutations often lead to a gain of
function. Mutations in one allele may also lead to a structural change in the
protein that interferes with the function of the wild-type protein encoded by
the other allele. These are referred to as dominant negative mutations.
Mutations can be advantageous and lead to an evolutionary advantage,
and can also be deleterious, causing disease, structural abnormalities, devel-
opmental delays, or other effects. In this chapter mutations occurring on a
small scale, such as substitutions, deletions, insertions, and exon skipping
resulting from mutations in splicing site are presented.

4.4.1 Substitution
Mutation called substitution occurs when one or more nucleotides are re-
placed by the same number of other nucleotides. In most cases it is only one
nucleotide (a point mutation). One purine, i.e. A or G, could be replaced
with another purine or one pyrimidine, i.e. C or T, with another pyrimidine
(such substitution is called a transition). Replacement of a purine with a
pyrimidine or vice versa is called a transversion. Transition mutations are
about an order of magnitude more common than transversions. Because of
the substitution consequences, they can be divided into silent mutations (not
leading to changes in the encoded protein), mutations leading to amino acid
change in the encoded protein (missense), and mutations inserting premature
stop codon (nonsense) (Fig. 4.9).

(a) (b)

Fig. 4.9 Categorizing point mutations: (a) illustration of transition (blue arrows)
and transversion (red arrows), (b) functional categorization: examples of silent mu-
tation, mutation leading to amino acid change in the encoded protein, and mutation
introducing a premature stop codon
58 4 DNA Replication, Mutations, and Repair

4.4.2 Deletion and Insertion

Deletion is a mutation in which one or more nucleotides are removed from
the DNA sequence. When three nucleotides are deleted (equivalent of the
codon) one amino acid is lost in the protein chain, but the rest is not
changed (Fig. 4.10a). The deletion which is not a multiple of three nucleotides
(Fig. 4.10b, c, d) leads to shift of the reading frame and change of all codons
starting from place of deletion (ch. 6.1.4).

(a) (b)

(c) (d)

Fig. 4.10 Examples of deletions leading to the development of the disease: (a)
deletion in the F9 gene that could be connected with hemophilia B; (b) and
(c) deletions in the CFTR gene that can cause genetic disorders (the blockage
of the ions and water movement into and out of the cell); (d) deletion in the
APC gene that could occur early in cancers, such as colon cancer. Sourced from:
http://www.web-books.com/MoBio/Free/Ch7E.htm

Insertion consists in insertion of one or more extra nucleotides into the

sequence. Similarly to deletion it can cause frameshift and formation of ab-
normal protein or, if inserted nucleotides are a multiple of three, new amino
acids are added. Insertions which does not change reading frame also can
lead to serious diseases (Table 4.1). Deletions and insertions often occur in
repetitive sequences.

4.4.3 Exon Skipping

Cutting of intron requires the presence of “GU . . . . . . . . . AG” signal (Fig. 4.11;
ch. 5.8.4). If the splicing acceptor site AG is mutated (e.g. A to C),
4.4 Mutations and Their Consequences 59

Table 4.1 Examples of diseases caused by insertions

Affected Repetitive Number of repeats Direct consequence

Disease gene sequence normal mutated of mutation
methylation of the
Fragile X syndrome fragile X mental CGG 6-44 > 200 promoter leading to
(common cause of autism retardation 1 the silencing of the
and intellectual disability) (FMR1 ) promoter FMR1 gene
Huntington’s disease (neu- expansion of the
rodegenerative disorder) huntingtin (HTT ) CAG 9-35 37-100 polyglutamine tract
Spinal and bulbar muscular
atrophy (SBMA) (neuro- androgen receptor CAG 7-24 40-55 expansion of the
degenerative disorder) (AR) polyglutamine tract
Spinocerebellar ataxia
(SCA1) (neurodegenera- ataxin 1 CAG 19-36 43-81 expansion of the
tive disorder) (ATXN1 ) polyglutamine tract
Dentatorubral-pallidoluysian
atrophy (DRPLA) (spinoce- atrophin 1 CAG 7-23 > 49 expansion of the
rebellar degeneration) (ATN1 ) polyglutamine tract
Duchenne muscular dystrophin premature translation
dystrophy (DMD) CTG 5-35 50-4,000 termination

Fig. 4.11 Illustration of exon skipping

ribonucleoprotein complex cutting intron will search for the next acceptor
place, which will result in excision of exon together with introns.

4.4.4 The Relationship between Mutations and

Polymorphisms
DNA sequence variations are sometimes described as mutations and someti-
mes as polymorphisms. A mutation could be defined as any change in a DNA
sequence away from normal. In contrast, a polymorphism is a DNA sequ-
ence variation that is common in the population. The arbitrary cut-off point
between a mutation and a polymorphism is 1%. That is, to be classed as a po-
lymorphism, the least common allele must have a frequency of 1% or more in
the population. If the frequency is lower, the allele is regarded as a mutation.
60 4 DNA Replication, Mutations, and Repair

The most common type of variation in the human genome is the single
nucleotide polymorphism (SNP or “snip”), where a single base differs be-
tween individuals. SNPs occur about once every 1,000 — 2,000 base pairs
in the genome. Polymorphic sequence variants usually do not directly cause
diseases. Many are completely neutral in effect, some may influence charac-
teristics such as height and hair colour rather than characteristics of medical
importance. However, polymorphic sequence variation does contribute to dis-
ease susceptibility and can also influence drug responses.

4.5 Mutation Mechanisms

Mutations can be caused by external (UV light, chemical agents, viruses, etc.)
or endogenous (toxic products of cellular metabolism) factors, or they may
be caused by errors in the cellular machinery (replication errors, accidental
deamination, etc.).
Deamination (Fig. 4.12) of cytosine to uracil in DNA is potentially muta-
genic because uracil forms a pair with adenine. After replication, one of the
daughter strands will contain the modified pair A:U instead of the correct
pair G:C. However, since uracil is not the part of DNA, this mutation can
easily be detected and repaired by base excision (ch. 4.6.1). Althought the
chemical structure of uracil is simpler than the structure of thymine, in DNA
thymine is used, not uracil. Otherwise, deamination of cytosine to uracil could
be distinguished from properly inserted uracil only by the incorrect pairing
with the complementary base. It would have to be corrected in the process of
mismatch repair (ch. 4.6.3), which is much less effective than base excision.
Thus, thymine is present in DNA because it increases the reliability of the
genetic code. Unlike DNA, RNA can not be repaired, so uracil is used to
RNA synthesis as “cheaper” element (its synthesis requires less energy than
synthesis of thymine).

Fig. 4.12 Examples of deamination, i.e. the removal of the amine group. Accidental
deamination can change the cytosine to uracil or the methylated cytosine to thymine.
4.5 Mutation Mechanisms 61

Spontaneous deamination of 5-methylcytosine results in thymine (Fig. 4.12).

This is the most common single nucleotide mutation in DNA. The repair mech-
anisms do not recognize thymine as erroneous, thus this change cannot be
corrected. It coud be a reason for the rarity of CpG sites (ch. 4.5.4) in the
eukaryotic genome.

4.5.1 Mutations Caused by UV Light

When DNA is exposed to UV light, two neighboring pyrimidines (cytosines
or thymines) may form a dimer (Fig. 4.13). Fortunately, most of these ge-
netic lesions are corrected seconds after they are created, before they can do
permanent damage. However, unrepaired dimers are mutagenic (Fig. 4.14).

Fig. 4.13 Thymidine dimers induced by UV light (similar dimers may be formed
from cytosine)

Fig. 4.14 Possible mechanism of mutation induced by UV light. If the cytosine

dimer is formed by UV light, during replication in the new strand (light blue) ade-
nine could be incorporated (instead of guanine). In the next round of replication
opposite to the adenine, thymine will be built, which will cause the mutation con-
sisting in a change of CC to TT. While cytosine dimers can be repaired, mutations
arising after replication is no longer detectable by the DNA repair system.
62 4 DNA Replication, Mutations, and Repair

4.5.2 Mutations Caused by Chemical Agents

Chemical agents that induce mutations in DNA are called mutagens and are
said to be mutagenic. Most of them are also carcinogens. In figures 4.15 and
4.16 structures of several potential mutagens and the mechanism of mutation
induced by some of them are shown.

Fig. 4.15 Examples of potential mutagens: I. Nitrous acid is a deaminating factor,

which converts cytosine to uracil, adenine to hypoxanthine, guanine to xanthine;
these bases have a different potential to form hydrogen bonds than the initial ones,
which leads to changes in the pairing of bases; II. Alkylating agents which add
alkyl group (-Cn H2n +1 ) to other molecules; nucleobase alkylation in DNA may
alter normal base pairing and thus cause mutations; some alkylating agents can also
cross-link DNA molecules, causing breaks in chromosome; III. A base analog is a
chemical that can substitute for a normal nucleobase in nucleic acids (5-bromouracil
— for thymine, 2-aminopurine — for adenine); IV. Acridines (e.g. proflavine) are
positively charged molecules; they can be inserted between two strands of DNA,
which leads to disorders in DNA structure and replication; V. Hydroxylamine and
free radicals alter the structure of bases, leading to changes in the base pairing.
4.5 Mutation Mechanisms 63

4.5.3 Mutations Caused by Replication Errors

Errors are natural part of the DNA replication. Although cells employ an
arsenal of editing mechanisms to correct mistakes, they do happen. They are
the main source of mutations. The frequency of mutations that have not been
corrected at the time of replication is one mutation per one cell division.

(a)

(b)

Fig. 4.16 (a) Structures of nitrogenous bases formed under the influence of free
radicals; (b) change of base induced by hydroxylamine (NH2 OH)

Most DNA replication errors are caused by mispairings of a different

nature. For example in repetitive sequences, where a new strand may be
incorrectly paired with the template strand, the replication slippage is com-
mon (Fig. 4.17). Such shifts formed during replication lead to the creation of
polymorphisms, e.g. in the microsatellite sequences containing thousands of
repetitive sequences. If the mutation occurs in a protein-coding sequence, it
can lead to production of incorrect protein and cause a disease. Huntington’s
disease is a well known example (Table 4.1).

4.5.4 DNA Methylation and CpG (CG) Islands

The CG island is a genomic region, in which the frequency of the CG se-
quence is higher than in other regions. It is also called the CpG island, where
“p” means that C and G are linked with each other by a phosphodiester
bond. In vertebrates, CpG islands are often found in the promoters of genes
64 4 DNA Replication, Mutations, and Repair

(a)

(b)

(c)

Fig. 4.17 Mutations caused by replication slippage within the repetitive sequence.
In this figure wrong pairing involves only one repetition but there could be more
of them. (a) Normal replication; (b) backward slippage, resulting in the insertion
mutation; (c) forward slippage, resulting in the deletion mutation.

required for the maintenance of basic cellular function (so-called housekeep-

ing genes), or genes that are frequently expressed. In the promoters of active
genes, CpG sequences are not methylated, whereas in inactive genes - they
are usually methylated, what is the reason of gene expression inhibition. The
cytosines in the CpG tend to be methylated. This methylation helps to dis-
tinguish the newly synthesized DNA strand from the parental one, which is
important in the final stages of DNA proofreading after duplication. How-
ever, methylated cytosine can be converted into thymine by the accidental
deamination (Fig. 4.12). Such mutation can only be corrected by mismatch
repair, which is very inefficient. Thus, over evolutionary time the methylated
CG sequences tend to turn into the TG sequences, which explains the lack
of CG sequences in inactive genes.
4.6 Mechanisms of DNA Repair 65

Each cell type has its own DNA methylation pattern, so that a unique set
of proteins can be produced in the cell and enable it to perform functions
specific for the cell type. During cell division, the DNA methylation pattern
passes to the daughter cell. This happens due to DNA methyltransferase,
which methylates only those CG sequences, which are paired with methylated
CG (Fig. 4.18).

Fig. 4.18 Inheritance of the DNA methylation pattern. DNA methyltransferase

methylates only those CpG sequences, which are paired with methylated CpG in
parental strand. Thanks to this, after replication of DNA the original methylation
pattern is preserved.

4.6 Mechanisms of DNA Repair

Most changes in DNA are quickly repaired. Those that are not repaired re-
sult in a mutation. Thus, mutation is, in fact, a consequence of the failure
of DNA repair. There are three main repairing mechanisms of damaged or
inappropriate bases: by base excision, by nucleotide excision, and by mis-
match repair. Other harmful changes in DNA, like double-strand breaks, are
repaired by non-homologous end joining (NHEJ) or homologous recombina-
tion. In NHEJ pathway, the broken ends are directly ligated without the
need for a homologous template, in contrast to homologous recombination,
which requires a homologous sequence to guide repair. Inappropriate NHEJ
66 4 DNA Replication, Mutations, and Repair

can lead to translocations and telomere fusion. Homologous recombination,

beside DNA repairing, produces also a new combinations of DNA sequences
during meiosis (ch. 7.4.1), the process by which eukaryotes make gametes,
like sperm and egg in animals.

4.6.1 Base Excision Repair (BER)

Nucleobases in DNA can be modified by deamination or alkylation. Damaged
or inappropriate base is recognized by DNA glycosylase, which removes it,
forming AP site (apurinic/apyrimidinic site). The backbone of the DNA
remains intact. AP site is then cleaved by an AP endonuclease. The resulting
single-strand break can then be processed by either short-patch or long-patch
base excision repair (Fig. 4.19).

Fig. 4.19 Basics steps of base excision repair: (a) long patch BER (2-10 nucleotide
patch); (b) short patch BER (one nucleotide patch)
4.6 Mechanisms of DNA Repair 67

4.6.2 Nucleotide Excision Repair (NER)

While the base excision repair machinery can recognize and correct only dam-
aged bases, the nucleotide excision repair (NER) enzymes recognize bulky
distortions in the shape of the DNA double helix. NER is a particularly
important for removing of the UV-induced DNA damage (e.g. thymidine
dimer). It is recognized differently depending on whether the DNA is tran-
scriptionally active (transcription-coupled repair) or not (global excision re-
pair) (Fig. 4.20). After the initial recognition step, the damage is repaired in
a similar manner. A short single-stranded DNA segment (24-32 nucleotides),
that includes the lesion, is removed. A single-strand gap created in the DNA is
subsequently filled in by DNA polymerase, which uses the undamaged strand
as a template.

Fig. 4.20 DNA repair by nucleotide excision. NER can be divided into two sub-
pathways that differ only in their recognition of helix-distorting DNA damage: (a)
transcription coupled NER and (b) global genomic NER.
68 4 DNA Replication, Mutations, and Repair

4.6.3 DNA Mismatch Repair (MMR)

The specificity of mismatch repair is primarily for base-base mismatches and
insertion/deletion mispairs generated during DNA replication and recom-
bination. The MMR machinery distinguishes the newly synthesised strand
from the template (which is treated as a correct one). In eukaryotes, the ex-
act mechanism for this is not clear. In gram-negative bacteria, a transient
hemimethylation distinguishes the strands: immediately after synthesis only
the template strand is methylated. In E. coli all adenines which in the tem-
plate strand are in the context of the sequence 5’-GATC-3’ are methylated by
a special methylase (Dam methylase). The enzyme that removes mismatch
cuts unmethylated strand, leaving the parental strand intact, so it could serve
as a template during the repair of DNA (Fig. 4.21).

Fig. 4.21 The mismatch repair in E. coli

The process begins with the binding of MutS protein within the wrong
paired base pairs. Then the MutL protein joins, which leads to binding and
activation of MutH. It cuts unmethylated strand within the sequence GATC.
The sequence from the place of incision to the wrong paired region is removed
by exonuclease with assistance of helicase II. The exonuclease recruited is
dependent on which side of the mismatch MutH incises the strand — 5’
or 3’. If the nick is on the 5’ end of the mismatch, either RecJ or ExoVII
(both 5’ to 3’ exonucleases) is used. If however the nick is on the 3’ end
of the mismatch, ExoI (a 3’ to 5’ enzyme) is used. Formed gap is filled by
References 69

DNA polymerase III and DNA ligase with assistance of SSB (single stranded
DNA-binding) proteins.
The distance between the GATC site and the area where there are mist-
akes in pairing rules could be up to 1,000 bp. Therefore, the mismatch repair
system is expensive and not very efficient. This system works similarly in
eukaryotic organisms. Proteins homologous to bacterial MutS and MutL were
found in yeast, mammals, and other organisms. These are the MSH5 MSH1
(homologous to MutS) and MLH1, PMS1, and PMS2 (homologous to MutL).
Mutations in the MSH2, PMS1, and PMS2 genes are associated with an
increased risk of colon cancer.

References
1. Berg, J.M., Tymoczko, J.L., Stryer, L.: DNA Replication, Recombination, and
Repair. In: Biochemistry, 5th edn. W.H. Freeman, New York (2002)
2. Clancy, S.: DNA damage & repair: mechanisms for maintaining DNA integrity.
Nature Education 1(1) (2008), http://www.nature.com/scitable/topicpage/
dna-damage-repair-mechanisms-for-maintaining-dna-344
3. King, M.W.: DNA Metabolism. In: themedicalbiochemistrypage.org, LLC (1996-
2012), http://themedicalbiochemistrypage.org/dna.php
4. Lodish, H., Berk, A., Zipursky, S.L., et al.: DNA Replication, Repair, and Re-
combination. In: Molecular Cell Biology, 4th edn. W.H. Freeman, New York
(2000)
5. Loewe, L.: Genetic mutation. Nature Education 1(1) (2008),
http://www.nature.com/scitable/topicpage/genetic-mutation-1127
6. Pray, L.: DNA replication and causes of mutation. Nature Education 1(1) (2008),
http://www.nature.com/scitable/topicpage/
dna-replication-and-causes-of-mutation-409
7. Pray, L.: Major molecular events of DNA replication. Nature Education 1(1)
(2008), http://www.nature.com/scitable/topicpage/
major-molecular-events-of-dna-replication-413
8. Pray, L.: Semi-conservative DNA replication: Meselson and Stahl. Nature Edu-
cation 1(1) (2008), http://www.nature.com/scitable/topicpage/
semi-conservative-dna-replication-meselson-and-stahl-421
9. Zvereva, M.I., Shcherbakova, D.M., Dontsova, O.A.: Telomerase: structure, func-
tions, and activity regulation. Biochemistry (Mosc) 75, 1563–1583 (2010)
Chapter 5
Transcription and Posttranscriptional
Processes

5.1 Gene Expression Overview

In multicellular organisms all somatic cells have the same set of DNA, yet
different cell types differ in shape and performed function. This is possible be-
cause different cells within the body express different portions of their DNA,
what determines diversity. Genes do not have influence on cellular functions
as long as they are not expressed.
Gene expression is the process by which information from a gene is used
in the synthesis of a functional gene product (a protein or functional RNA).
The process involves several steps (Fig. 5.1):
– transcription: on DNA template complementary RNA is synthesized (pre-
cursor RNA, pre-RNA);

Fig. 5.1 Subsequent steps of the expression of genes encoding proteins in eukary-
otic cells, where transcription and translation are separated in time and space. In
prokaryotes translation can begin before the end of gene transcription.

W. Widłak: Molecular Biology, LNBI 8248, pp. 71–92, 2013.


c Springer-Verlag Berlin Heidelberg 2013
72 5 Transcription and Posttranscriptional Processes

– RNA processing: modifications of pre-mRNA leading to the formation of

mature mRNA or a functional tRNA, rRNA, or ncRNA. In the case of
genes encoding RNA their expression ends when a functional RNA
molecules reach their destinations.
Expression of genes encoding proteins includes two additional steps:
– transport of mRNA to the cytoplasm;
– protein synthesis: in the cytoplasm mRNA binds to ribosomes where pro-
tein is synthesized on the basis of mRNA sequence.
According to the process described above, the flow of genetic information
occurs in the following direction: DNA → RNA → protein. This rule has
been called the central dogma of molecular biology because it was thought
that the same rule would apply to all organisms. However, it is not true for
RNA viruses, where the genetic information can be transferred back from the
RNA to the DNA.

5.2 Gene Transcription and RNA Polymerases

Transcription is a process in which one DNA strand is used as a template for
the synthesis of a complementary RNA. For example:

Thus, synthesized RNA strand has a nucleotide sequence complementary

to the DNA template strand and identical with the DNA coding strand,
except that in RNA instead of thymine (T) uracil (U) is present. The coding
strand of DNA is also called sense strand, plus strand, or non-template strand.
The template strand is called antisense strand, minus strand, or transcribed
strand. The nucleic acid strand is always synthesized in the 5’ to 3’ direction,
and the template is read in the 3’ to 5’ direction (Fig. 5.2). The reaction
is catalyzed by enzymes called polymerases: RNA is synthesized by RNA
polymerase.
In all species transcription begins with the binding of the RNA polymerase
to DNA at the transcription initiation site. Prokaryotic polymerases recognize
this site and bind directly to it, while eukaryotic polymerases are dependent
on other proteins called transcription factors (TFs). In eukaryotes, the
DNA sequence that determines the transcription start site is called a core
promoter. A fundamental step of transcription is unwinding of the DNA
fragment. The enzyme which unwinds the double helix is called a helicase.
Prokaryotic polymerases possess also the helicase activity. In eukaryotes DNA
is unwinded by a specific transcription factor (TFIIH; ch. 5.5).
5.2 Gene Transcription and RNA Polymerases 73

(a)

(b)

Fig. 5.2 Scheme illustrating the process of transcription: (a) DNA before the tran-
scription; (b) during the transcription fragment of DNA is unwinded, so that one
of the strand can be used as a template for the synthesis of a complementary RNA
(so-called transcription bubble is formed)

Activated RNA polymerase moves along the template and adds matching
RNA nucleotides that are paired with complementary DNA bases. Elongation
involves a proofreading mechanism that can replace incorrectly incorporated
bases. Transcript elongation leads to clearing of the promoter, and the tran-
scription process can begin yet again. Thus, multiple RNA polymerases can
work simultaneously on a single DNA template, and many RNA molecules
(especially mRNA) can be rapidly produced from a single copy of a gene.
RNA polymerase remains connected with DNA until it encounters on tran-
scription termination signal. Prokaryotic and eukaryotic organisms have dif-
ferent signals for transcription termination. (Note: the signal “stop”, which
is present in the genetic code, is a signal to terminate protein synthesis, not
transcription). Transcription in eukaryotic cells is much more complicated
than in prokaryotes, partially because the eukaryotic DNA is associated with
histone proteins that may block the access of polymerase to the promoter.

5.2.1 RNA Polymerases

Both RNA and DNA polymerases are enzymes that add nucleotides to an
existing strand of nucleic acid, what leads to its elongation. However, RNA
polymerase can start a new strand synthesis without any primer.
RNA polymerase uses nucleoside triphosphates (NTPs). Synthesis starts from
pppG or pppA (p — phosphate group). Nucleotide is attached to the RNA,
while two phosphate groups in the form of pyrophosphate (PPi ) are released
74 5 Transcription and Posttranscriptional Processes

Fig. 5.3 Chemical reaction catalyzed by RNA polymerase

(Fig. 5.3). Nucleic acid strand is always synthesized in the 5’ to 3’

direction.
Prokaryotic (e.g. E. coli) RNA polymerase is a large (approximately
500 kDa) enzyme complex consisting of five subunits: four catalytic (two
α, one β, one β  ) and a single regulatory (σ). Beta subunits (β, 151 kD, and
β  , 156 kD) are much larger than α subunit (37 kD). Sigma subunit (also
called σ factor) appears in several forms, with a mass from 28 kD to 70 kD.
It plays an important role in recognizing the transcription initiation site and
also shows helicase activity. Catalytic subunits (α2 ββ  ) form polymerase
core. Together with σ subunit it is called holoenzyme. The term “holoen-
zyme” is generally applied to enzymes that contain multiple protein subunits,
all needed for activity.
5.3 DNA Regulatory Elements 75

In eukaryotes there are three classes of RNA polymerases: I, II and III.

Each of them consists of two large and 12 to 15 smaller subunits. Large sub-
units are homologs of prokaryotic β and β  subunits, and two smaller are
similar to the α subunit of E. coli. Eukaryotic polymerases do not contain
subunits corresponding to σ factor in E. coli. In eukaryotes other proteins
are involved in the initiation of transcription.
Polymerase II is the most important among the eukaryotic RNA poly-
merases. It is involved in the transcription of protein-coding genes, snRNA
and miRNA. The other two polymerases are responsible only for the tran-
scription of genes encoding RNA. RNA polymerase I is located in the nuc-
leolus and it synthesizes rRNA (except 5S rRNA). RNA polymerase III is
located outside the nucleolus and it synthesizes 5S rRNA, tRNA, U6 snRNA
and some small RNA.

5.3 DNA Regulatory Elements

In the simplest term, a gene is a region of transcription together with a regu-
latory region (Fig. 5.4). Transcription unit is a sequence of DNA undergoing
transcription (i.e., a sequence which serves as a template for complementary
primary transcript). In the regulatory region, cis-regulatory elements can
be distinguished. These are specific DNA sequences, to which transcription
factors (TFs) may bind. TFs are proteins, which after binding to DNA af-
fect transcription (enchance or suppress it). DNA sequences that encode
transcription factors are called trans-regulatory elements.

Fig. 5.4 The structure of a typical gene coding for protein in eukaryotes. Dark
rectangles within the transcription unit - coding sequences; bright rectangles - se-
quences coding for untranslated regions (UTRs) present in the 5’ and 3’ ends of the
mature transcript. Nucleotides in the sequence downstream from the transcription
start site (indicated by an arrow pointing to the right) are numbered strating from
+1, and upstream - from -1 (no 0 position).

With the regulatory region the terms “basic (core) promoter”, “enhancer”,
and “silencer” are also linked. The core promoter is formed from DNA seq-
uences determining transcription start site (abbreviated to TSS; also called
Inr — initiator). The enhancer is a sequence, which after binding of the
transcription factor (in this case — the activator) increases the activity of
76 5 Transcription and Posttranscriptional Processes

the promoter. The silencer, after binding of the transcription factor (re-
pressor), inhibits transcription. Sometimes the same sequence can enhance
or inhibit transcription, depending on the binding protein. Enhancers and si-
lencers could be found upstream or downstream from the transcription unit,
or even in introns.
In eukaryotes the most common element of the promoter of protein-coding
genes is the TATA box, found 20-35 bp upstream from the transcription
start site (at position -35 to -20). TATA box is nothing more than a se-
quence TATAAA (sometimes: TATATAT or TATATAA). The TATA box is
the binding site for a transcription factor known as TATA-binding protein
(TBP)(ch. 5.5). Upstream by 200 bases to the TSS, regulatory elements like
CAAT box or GC box commonly occur (Table 5.1).

Table 5.1 Sequences commonly found in promoters of eukaryotic genes and tran-
scription factors that bind to them

Name of the sequence Position Transcription factors Consensus sequence

TATA box -35 to -20 TATA-box binding protein (TBP) TATAAA
CAAT box -110 to -50 CAAT binding protein (CBF), GGCCAATCT
nuclear factor 1 (NF1), or
CAAT/enhancer binding
protein (C/EBP )
GC box -200 to -70 Specificity protein/Krüppel-like GGGCGG
factor (SP/KLF) family

5.3.1 Response Elements

The DNA sequence recognized by a specific transcription factor in response
to some stimuli is called the response element. Most response elements are
located within one thousand bp above the TSS. Transcription factors inter-
act with their binding sites using a combination of electrostatic and Van der
Waals forces. Due to the nature of these chemical interactions, most transcrip-
tion factors bind DNA in a sequence specific manner. However, transcription
factors do not bind just one sequence but are capable of binding a subset of
closely related sequences, each with a different strength of interaction. The
most common sequence recognized by a given transcription factor is called
consensus, or logo sequence.
Table 5.2 describes some of the eukaryotic response elements. CRE (cAMP
response element) interacts with CREB (CRE-binding) protein, which is reg-
ulated by cAMP (ch. 8.5.1). ERE (estrogen response element), and GRE (glu-
cocorticoid response element) are identified respectively by estrogen and
glucocorticoid receptors. Estrogen and glucocorticoid are steroid hormones.
Although they are not transcription factors, they may affect gene expression
through their receptors (ch. 8.1).
5.4 Structural Motifs of Transcription Factors 77

Table 5.2 Examples of eukaryotic response elements and transcription factors that
bind to them
Name of the sequence Transcription factor Consensus sequence
cAMP response element
CRE binding protein (CREB) TGACGTCA
ERE Estrogen receptor (ER) AGGTCANNNTGACCT
GRE Glucocorticoid receptor (GR) AGAACANNNTGTTCT
SRE Serum response factor (SRF) CC(A/T)6 GG
HSE Heat shock factor 1 (HSF1) NTTCNNGAANNTTCN
(A/T)6 = six A or T; N = any base.

The serum response factor (SRF) activated by growth factors present in

serum binds to the serum response element (SRE). SREs are usually present
in the promoters of genes involved in cell cycle progression.
In response to cellular stress (e.g. elevated temperature) heat shock factor
(HSF1) is activated. It binds to the HSE (heat shock element) sequences and
stimulates the expression of heat shock proteins (HSPs), which enables cell
to survive in adverse conditions.

5.4 Structural Motifs of Transcription Factors

Transcription factors must have an ability to interact with DNA. This
could be done using several structural protein motifs found in the DNA
binding domains (DBDs): zinc finger, leucine zipper, helix-turn-helix, or
helix-loop-helix.

The zinc finger motif is elongated protein subunit consisting of 30 amino

acids. The structure is stabilized by interactions between the zinc ion and two
cysteines and two histidines from side chains. Arranged tandemly zinc fingers
recognize longer DNA sequences. One of the first described protein contain-
ing this motif was a transcription factor TFIIIA. To transcription factors
possessing zinc finger motif belong also: SP1 (binds to DNA in regions rich
in GC pairs), estrogen receptor (binds to ERE sequence – estrogen response
element), and others steroid hormone receptors. The name of many proteins
containing zinc finger motif starts with “ZFP” (from “zinc finger protein”).

Leucine zipper binds to DNA as a dimer (homodimer or heterodimer), just

like many other transcription factors. It is composed of two chains of α-helix
structure, twisted and held together by the hydrophobic interactions between
lysine residues. Because lysines are located every seventh amino acid residue,
all of them are situated on one side of each helix. Leucine zipper structure
recalls the Y letter. Lysines are present in the folded part (from the carboxy
78 5 Transcription and Posttranscriptional Processes

terminus), while “arms” (from the amino terminus) are formed from basic
amino acids. Such structure allows binding to DNA. The arms can be folded,
allowing them to fit into the major groove of DNA. Structures of leucine
zipper have been found for example in AP-1 (activator protein 1; heterodimer
formed by JUN and FOS proteins) and CREB (ch. 8.5.1).

Helix-turn-helix is a structure containing two α-helices and a short ex-

tended chain of amino acids between them. Helix closer to the C-terminus
of the protein binds to the major groove of DNA. This motif is present in
thousands of proteins binding to DNA. A special variation of the motif is the
homeodomain, which is encoded by homeobox genes, playing a key role in
the development of the organism.

Helix-loop-helix is formed by two α-helices connected by a loop. Struc-

turally the motif is completely different from motif helix-turn-helix – it is
more like leucine zipper. Transcription factors containing helix-loop-helix do-
main normally function as heterodimers. An example of a transcription factor
with such a structure is MyoD (myoblast determination protein).

5.5 Transcription of Protein-Coding Genes

Eukaryotic RNA polymerase II does not have a subunit similar to the prokary-
otic σ subunit, which recognizes the promoter and unwinds the DNA double
helix. In eukaryotes these functions are performed by proteins called general
transcription factors (GTFs). RNA polymerase II can interact with six of
such factors, marked as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH
(TF - transcription factor, II - for the RNA polymerase II). Most of them have
a complex structure composed of multiple subunits. The exact order of gen-
eral transcription factors attachment to the promoter is not known, especially
as it can be different for different promoters. In some cases, preinitiation
complex, containing the general transcription factors and the polymerase,
is made independently of the DNA and then it binds to DNA. But usually
the process of mounting of the complex to the DNA occurs in a few steps
(Fig. 5.5).
In the first step of transcription initiation, TFIID factor associates with
TATA box sequence in the core promoter. TFIID is composed of two subunits:
TBP (TATA-box binding protein) and TAF (TBP-associated factor). TAF
assists TBP in binding to DNA. Some promoters do not have TATA sequence.
In this case, TAF binds to DNA first, and then forces TBP binding. In human
cells TAF is composed of 12 subunits. One of them can catalyse histones ac-
etylation, which leads to a loosening of interactions between histones and
DNA within a nucleosome (nucleosome can be moved and the DNA sequence
is accessible for TFs).
5.5 Transcription of Protein-Coding Genes 79

Fig. 5.5 Initiation of transcription in eukaryotes: (a) in most promoters TATA

box sequence is present, located about 25 bp upstream the transcription start site;
(b) TFIID binds at the place of TATA box; (c) connection of TFIIA stabilizes the
complex. TFIIB binds to DNA between TFIID and the future location of the RNA
polymerase II; (d) remaining general transcription factors and RNA polymerase
II bind to the promoter; (e) TFIIH, using energy from ATP, unwinds DNA at the
place of transcription initiation site, which allows starting of RNA synthesis. TFIIH
also phosphorylates the polymerase, which leads to the general transcription factors
releasing and the beginning of elongation.

After assembly of the preinitiation complex, TFIIH factor unwinds the DNA
starting at the position of about -10. Transcription bubble is formed and RNA
polymerase II begins synthesis. However, usually only a low (a basal) rate of
transcription is driven by the preinitiation complex alone. For modulation of
the transcription rate activators and repressors (along with any associated
coactivators or corepressors) are responsible (ch. 5.7). During RNA elongation,
TFIIF remains associated with the polymerase, while other transcription fac-
tors dissociates. Polymerase must be phosphorylated during the elongation.
Elongation of RNA chain proceeds as long as the polymerase encounters
a “stop” signal for the transcription. Synthesis of some eukaryotic RNAs is
finished in a manner similar to transcription termination in prokaryotes, that
is by creating a hairpin structure (containing arm and loop), followed by the
U bases. But ussually eukaryotes use different signals to terminate transcrip-
tion than prokaryotes. The termination mechanism is not exactly known.
80 5 Transcription and Posttranscriptional Processes

Eukaryotic protein-coding genes contain a polyadenylation signal, and often

transcription ends in 500-2,000 bp downstream of this signal. Finally, dur-
ing the RNA processing, sequence downstream of polyadentlation signal is
removed (ch. 5.8.2).

5.6 Transcription of RNA Genes

Transcription of genes coding for functional RNA molecules (rRNA, tRNA
and small RNA) seems to be less complex than protein-coding genes. In ma-
mmals three the rRNA genes (28S, 18S, and 5.8S) are grouped together as
a pre-rRNA gene. It is transcribed by RNA polymerase I. tRNA, 5S rRNA,
and U6 snRNA are transcribed by RNA polymerase III, while most snRNA
genes are transcribed by RNA polymerase II (just like protein-coding genes).
Preinitiation complex formed during the pre-rRNA synthesis is composed
of two upstream binding factors (UBFs). They bind to DNA forming a loop
between the sequence directly preceding the transcription start site (core
element) and upstream control element (UCE). Then TBP (TATA-binding
protein), TAFIs (TBP-associated factors), and RNA polymerase I enter the
complex, which allows to start the transcription (Fig. 5.6).

Fig. 5.6 Mounting of the preinitiation complex for RNA polymerase I

5.7 The Role of Transcription Factors and Chromatin Structure in Regulation 81

Genes encoding tRNA have two regulatory elements (A box and B box)
located within the transcription unit. Mounting of the preinitiation complex
begins with binding of TFIIIC to these elements. Transcription of the gene
encoding the 5S rRNA also depends on regulatory element located within the
transcription unit (C box). First TFIIIA binds to it, than, TFIIIC. Joining
of further proteins, necessary to start the synthesis of tRNA and 5S rRNA, is
carried out analogically: TBP protein enters the complex, then BRF, B" and
RNA polymerase III (Fig. 5.7). TBP protein (TATA-binding protein) is nec-
essary to create a preinitiation complex for each of three RNA polymerases.

(a) (b)

Fig. 5.7 Mounting of the preinitiation complex for RNA polymerase III: (a) tRNA
gene transcription; (b) 5S rRNA gene transcription

5.7 The Role of Transcription Factors and Chromatin

Structure in Regulation of Transcription
Assembly of the preinitiation complex from the general transcription fac-
tors and RNA polymerase II is usually not enough to start the transcription
process. With proteins gathered around the transcription start site, another
transcription factors (activators) interact. They could be bound to a cis-
regulatory elements located far away from the core promoter (Fig. 5.8). RNA
molecules are also involved in the regulation of many genes expression.
82 5 Transcription and Posttranscriptional Processes

Fig. 5.8 The interactions between transcription factors and changes in chromatin
structure during activation of transcription

Gene expression is also regulated by so called looping factors and other

interacting factors (coactivators), such as histone acetyltransferases (HATs).
Their task is remodeling of chromatin structure. Their presence is usually
necessary to enhance transcription by activators. Catalyzed by HAT acetyla-
tion of lysine residues neutralizes the positive charge on the histones, thereby
decreasing the interaction of the N termini of histones with the negatively
charged phosphate groups of DNA. As a consequence, the condensed chro-
matin is transformed into a more relaxed structure that is associated with
greater levels of gene transcription. This relaxation can be reversed by histone
deacetylase (HDAC) activity. One of the most important acetyltransferases
are the CBP protein (CREB Binding Protein), and highly related to it, p300
protein. Both proteins form a family of the transcription coactivators denoted
as CPB/p300.
An additional mechanism that regulates gene expression is DNA methy-
lation, which is attachment of methyl groups (-CH3 ) to cytosines in DNA
chain (Fig. 2.10). Methylation of CpG islands in the promoter is connected
with inhibition of the gene transcription. DNA methylation pattern in the
genome is reproduced after cell division (ch. 4.5.4). Thus, changes in chro-
matin that affect the expression of genes are inherited, although they are not
associated with changes in DNA sequence. The science describing such mech-
anisms is called epigenetics. In addition to DNA methylation, to elements of
epigenetic gene regulation also belong: modifications of core histones (phos-
phorylation, methylation, acetylation), the phenomenon of RNA interference
(RNAi) (ch. 3.3.4), and gene bookmarking.

5.7.1 Mechanism of Gene Bookmarking

Gene bookmarking is a mechanism of epigenetic memory. It enables to trans-
fer information to the daughter cells which genes have to be active and
which might be activated. This results in maintaining the phenotype (i.e.
the characteristic features) of a cell line. To create a gene bookmarks on the
entire pool of active genes, TFIID and TFIIB general transcription factors
5.7 The Role of Transcription Factors and Chromatin Structure in Regulation 83

(that form a basic preinitiation complex) most frequently are used. During
mitosis (cell division), they remain associated with the promoters of genes
that were active before the beginning of mitosis. At the same time core his-
tones are modified (Fig. 5.9). This prevents from the complete “packing” of
the DNA at these loci during mitosis. Just after mitosis (in early G1 phase),
binding of protein complex that triggers transcription is facilitated in marked
loci. Thus, the gene expression profile in cell remains unchanged.

Fig. 5.9 Model of creating gene bookmarks on the entire pool of transcription-
ally active genes. Altered from: Sarge K. D. and Park-Sarge O. K. (2005) Gene
bookmarking: keeping the pages open. Trends Biochem. Sci. 30, 605-610.

More specific bookmarks are created from proteins of Polycomb (PcG)

and Trithorax (TrxG) group, which regulate the expression of homeobox
genes, relevant for development of the organism. They interact with DNA
within so called cellular memory modules. Also HSF2 transcription factor
remains bound with promoter of Hsp70 genes during the cell division, which
keeps them in standby to start transcription immediately after the division,
if necessary.

5.7.2 Insulators
Eukaryotic transcriptional activators (enhancers) are able to function over
long distances and in an orientation independent manner. Gene expression
can be dependent also on chromatin structure. Both enhancers and condensed
chromatin could encroach on adjacent genomic domains to perturb gene
84 5 Transcription and Posttranscriptional Processes

expression. A class of DNA sequence elements that possess an ability to

protect genes from inappropriate signals emanating from their surrounding
environment are called insulators. They can block the action of a distal en-
hancer on a promoter (if the insulator is situated between the enhancer and
the promoter). Insulators protect genes also by acting as “barriers” that pre-
vent the advance of nearby condensed chromatin that might otherwise silence
expression. Some insulators are able to act both as enhancer blockers and bar-
riers. Others, particularly in yeast, serve primarily as barriers. All character-
ized enhancer-blocking elements identified in vertebrates bind CTCF protein
(CCCTC-binding factor).
In the laboratory, insulators could be used when constructing artificial
genes, which are later introduced into cells. The most known insulator is a
sequence from 5’ end of a chicken β-globin, which forms the boundary bet-
ween the “open” and “condensed” chromatin. Insulators help to preserve the
independent expression of genes located near the regulatory sequences, which
should be ignored.

5.8 RNA Processing

Post-transcriptional processing of RNA is necessary to obtain the mature
form of mRNA or functional tRNA and rRNA from the primary transcript.
In prokaryotes, mRNA molecules are modified only slightly or not at all.

Fig. 5.10 Scheme of mRNA processing in eukaryotes

5.8 RNA Processing 85

Many of them undergo translation before the end of transcription. Whereas

in eukaryotes, pre-mRNA must undergo post-transcriptional modifications
before it can be used in translation. Otherwise mRNA would be destroyed
in the cytosol by proteins which task is to destroy the nucleic acids. It is a
method of defense against intrusion into the cell of foreign nucleic acid, such
as viral.
Pre-mRNA processing includes the following steps (Fig. 5.10):
– capping - addition of guanylate cap, that is 7-methylguanosine (m7G), to
the 5’ end;
– polyadenylation - addition of the poly(A) tail to the 3’ end;
– splicing - removal of introns and connection of exons.
In some cases editing of RNA also occurs.

5.8.1 Capping Process

Addition of a structure called a cap occurs shortly after the start of transcrip-
tion. The first phosphate group at the 5’ end of the transcript is removed by
hydrolysis and in its place GTP (guanosine triphosphate) is added losing
two phosphate groups: a unique 5’-5’ triphosphate linkage is created. Then
7-nitrogen of guanine (N-7) is methylated. In most organisms, the first (and
often the second) nucleotide is also methylated at the 2’-hydroxyl of the ri-
bose (Fig. 5.11). Cap affects mRNA stability by protecting the 5’ end from
phosphatases and nucleases. It is also an important element in the transport
of mRNA to the cytoplasm: it interacts with proteins of the exporting com-
plex TREX (transcription export complex protein), which carries a transcript
by nuclear pores and brings it to ribosomes.

5.8.2 Polyadenylation
To the 3’ end of the transcript adenylate residues are added. In the first stage
of polyadenylation, the primary transcript is digested by specific endonu-
clease recognizing the sequence AAUAAA, which is polyadenylation signal.
Cutting occurs about 10 - 35 nucleotides below the sequence. A sequence rich
in GU (or U), located about 50 nt below, is an additional determinant of the
cleavage site. Next, poly(A) polymerase adds about 250 A residues (in yeast
- about 100) to the molecule of RNA (Fig. 5.12). Thanks to poly(A) tail
mRNA molecule is recognized as own, not foreign nucleic acid, and is pro-
tected against digestion by nucleases. Some viruses, such as influenza virus,
can attach to their mRNA poly(A) tail, and by that they are not destroyed
in the cytosol. Poly(A) tail also increases the effectiveness of translation.
86 5 Transcription and Posttranscriptional Processes

Fig. 5.11 Cap formation on the 5’ end of eukaryotic mRNA

Fig. 5.12 Scheme of polyadenylation of primary transcript in eukaryotes

5.8 RNA Processing 87

5.8.3 Processing of Pre-rRNA and Pre-tRNA

In mammals, the pre-rRNA transcript contains three rRNA: 18S, 5.8S and
28S. Transcription and processing occur in the nucleolus. U3 snRNA and
other U-rich snRNAs and proteins associated with them are involved in the
cutting of pre-rRNA. Synthesized in nucleoplasm 5S rRNA does not require
post-transcriptional processing. When it is finished, it moves to the nucleo-
lus and together with 28S rRNA and 5.8 S rRNA forms a large subunit of
ribosome.
In eukaryotes, tRNA precursors develop into mature molecules in a series
of changes involving:
– removal of the leader sequence (approximately 16 nt) from the 5’ end by
RNase P;
– removal of intron (approximately 14 nt) from the anticodon loop;
– replacement of two U residues at 3’ end of the molecule by CCA;
– modification of some bases.

5.8.4 RNA Splicing

In the process of splicing, introns are removed from primary transcript and
exons are joined together. Splicing must occur very precisely, because even
a single-nucleotide shift can move the reading frame and completely change
a sequence of amino acids. Well-defined signal for splicing is usually present
in introns. Most introns begin with GU sequence from the 5’ end (splice
donor site) and end with AG (splice acceptor site). Also the presence of the
CU(A/G)A(C/U) sequence is important (where A is preserved in all genes).
It is called a branch site and is located 20-50 nt upstream from the splice
acceptor site (Fig. 5.13). In 60% of cases exon has the sequence (A/C)AG in
the donor site, and G in the acceptor site. Moreover, auxiliary cis-elements,
known as exonic and intronic splicing enhancers (ESEs and ISEs) and exonic
and intronic splicing silencers (ESSs and ISSs), aid in the recognition of exons.

Fig. 5.13 The classical splicing signals found in the major class (>99%) of human
pre-mRNAs. Pu — purine (A or G), Py — pyrimidine (C or U). Py-rich — sequence
rich in pyrimidine. Auxiliary elements are not shown.
88 5 Transcription and Posttranscriptional Processes

Fig. 5.14 Scheme of spliceosome formed during intron removing. U1-U6 —

snRNA-protein complexes (snRNP).

Components of the basal splicing machinery bind to the classical splice-

site sequences and promote assembly of the multicomponent splicing com-
plex known as the spliceosome (Fig. 5.14). The spliceosome performs the
two primary functions of splicing: recognition of the intron/exon boundaries
and catalysis of the cut-and-paste reactions that remove introns and join
exons. The spliceosome is made up of five small nuclear ribonucleoproteins
(snRNPs) and more than one hundred proteins. Each snRNP is composed of
a single uridine-rich small nuclear RNA (snRNA) and multiple proteins. The
U1 snRNP binds the 5’ splice site, and the U2 snRNP binds the branch site
via RNA:RNA interactions between the snRNA and the pre-mRNA. Spliceo-
some assembly is highly dynamic. Both splice sites are recognized multiple
times by interactions with different components during the course of spliceo-
some assembly.
The typical human gene contains an average of 8 exons. Internal exons av-
erage 145 nucleotides (nt) in length, and introns average more than 10 times
this size and can be much larger. Most pre-mRNAs undergo alternative splic-
ing. In such cases some splicing signals are masked by the regulatory proteins.
Alternative splicing is a major mechanism for modulating the gene expression
of an organism. It enables a single gene to increase its coding capacity, allow-
ing the synthesis of several structurally and functionally distinct mRNA and
protein isoforms from a unique gene. However, disruption of normal splicing
patterns can cause or modify human diseases (e.g. muscular dystrophy).

5.8.5 RNA Editing

Information contained in some RNA molecules can be changed just after the
end of the transcription in a process different than splicing. Change of a single
nucleotide in mRNA may cause a codon change. For example deamination of
5.9 Reverse Transcription 89

cytosine in glutamine codon CAA leads to the formation of uracil and UAA
stop codon, and so, to earlier termination of translation as it is in apo-B gene
(Fig. 5.15). In mammals apo-B gene is expressed in liver and in epithelium
of small intestine. In liver, 500 kDa protein is created (called Apo-B100), in
intestine — much smaller (called Apo-B48). Apo-B100 is produced without
RNA editing, while Apo-B48 is produced from mRNA, in which cytosine in
CAA codon has been deaminated by an enzyme present in epithelial cells of
intestine (but not in liver).

Fig. 5.15 Editing of the apolipoprotein B RNA

5.9 Reverse Transcription

In RNA viruses (called retroviruses), RNA is the carrier of genetic informa-
tion. After entering a host cell, viral RNA is transcribed to a double-stranded
DNA (in a process called reverse transcription) and is integrated into the
host DNA. Reverse transcription is catalyzed by reverse transcriptase, an
enzyme which is introduced into the host cell by virus during infection. Re-
verse transcriptase catalyzes three types of reaction: synthesis of DNA on
RNA template, synthesis of DNA on DNA template, and RNA hydrolysis
(Fig. 5.16).
Transcription of retroviral DNA occurs only when it is integrated into the
DNA of the host (Fig. 5.17). Integration occurs thanks to IN protein (in-
tegration protein; integrase) which is introduced into the cell by the virus
together with the reverse transcriptase. IN is an endonuclease, which recog-
nizes specific sequences in LTR. Integration of viral DNA can occur in any
place in the host genome.
Genetic strategy used by retroviruses can be observed in various eukaryotic
genetic elements, for example in Ty elements found in yeast (T – transposon,
y – yeast). Ty1 is a transposon (i.e. mobile genetic element) of length of 6
kbp, integrated with the yeast genome. It has a border LTR sequences and
is flanked by short repeated sequences. Ty1 encodes proteins similar to the
90 5 Transcription and Posttranscriptional Processes

Fig. 5.16 The mechanism of reverse transcription of viral RNA. The process is
catalysed by reverse transcriptase — enzyme that has DNA polymerase and RNase
H activities. (1) Cellular tRNA specific for the retrovirus hybridizes with a com-
plementary region of the virus genome called the primer binding site (PBS). (2)
DNA synthesis begins from the 3’-OH group of attached tRNA on the template of
retrovirus genomic RNA. DNA fragment (-) complementary to the sequence of U5
and R is created. (3) The viral U5 and R sequences are removed by the enzyme
domain with RNase H activity. (4) First shift: DNA strand (-) hybridizes with the
R sequence present in the 3’ end of viral RNA. (5) DNA strand (-) is extended
from the 3’ end. (6) Most viral RNA is removed by RNase H. (7) The remaining
fragment of viral RNA is used as primer for synthesis of the second strand of DNA
(+). (8) The remaining viral RNA and tRNA are removed by RNase H. (9) Second
shift: PBS region of the second DNA strand (+) hybridizes with the PBS region of
the first DNA strand (-). (10) Synthesis of the missing parts of both strands. At the
ends of retroviral DNA, long terminal repeats, called LTR sequences, are formed.
U3 and U5 have different unique sequences, R is a repeated sequence. LTRs contain
signal sequences for transcription and integration processes.
References 91

Fig. 5.17 Formation of retroviral genomic RNA

retroviral reverse transcriptase and integrase. Duplication and transfer (trans-

position) to any other place in the genome occurs with the participation of
an intermediate product — RNA, which is produced in transcription process.
Then, on Ty RNA template, double-stranded DNA is synthesized, which is
included to the genome. So, the flow of Ty transposon information (from
DNA to RNA and back to the DNA) is the same as in retroviruses. Genetic
elements that use this strategy are called retrotransposons or retroposons
(ch. 7.4.3). Similar elements were found in Drosophila (copia elements).

References
1. Berg, J.M., Tymoczko, J.L., Stryer, L.: RNA Synthesis and Splicing. In: Bio-
chemistry, 5th edn. W. H. Freeman, New York (2002)
2. Clancy, S.: DNA transcription. Nature Education 1(1) (2008),
http://www.nature.com/scitable/topicpage/dna-transcription-426
3. Clancy, S.: RNA splicing: introns, exons and spliceosome. Nature Educa-
tion 1(1) (2008), http://www.nature.com/scitable/topicpage/
rna-splicing-introns-exons-and-spliceosome-12375
4. Hoopes, L. (ed.): Gene Expression and Regulation. In: Miko, I. (ed.) Genetics.
Nature Education (2011), http://www.nature.com/scitable/topic/
gene-expression-and-regulation-15
5. Katahira, J., Yoneda, Y.: Roles of the TREX complex in nuclear export of
mRNA. RNA Biol. 6, 149–152 (2009)
6. King, M.W.: Regulation of Gene Expression. In: themedicalbiochemistry-
page.org, LLC (1996-2012), http://themedicalbiochemistrypage.org/
gene-regulation.php
7. King, M.W.: RNA Transcription & Processing. In: themedicalbiochemistry-
page.org, LLC (1996-2012),
http://themedicalbiochemistrypage.org/rna.php
8. Lodish, H., Berk, A., Zipursky, S.L., et al.: Regulation of Transcription Initia-
tion. In: Molecular Cell Biology, 4th edn. W.H. Freeman, New York (2000)
9. Lodish, H., Berk, A., Zipursky, S.L., et al.: RNA Processing, Nuclear Transport,
and Post-Transcriptional Control. In: Molecular Cell Biology, 4th edn. W.H.
Freeman, New York (2000)
10. Proudfoot, N.J.: Ending the message: poly(A) signals then and now. Genes
Dev. 25, 1770–1782 (2001)
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11. Sarge, K.D., Park-Sarge, O.K.: Gene bookmarking: keeping the pages open.
Trends Biochem. Sci. 30, 605–610 (2005)
12. Vogelmann, J., Valeri, A., Guillou, E., et al.: Roles of chromatin insulator pro-
teins in higher-order chromatin organization and transcription regulation. Nu-
cleus 2, 358–369 (2011)
13. Ward, A.J., Cooper, T.A.: The pathobiology of splicing. J. Pathol. 220, 152–163
(2010)
14. Weth, O., Renkawitz, R.: CTCF function is modulated by neighboring DNA
binding factors. Biochem. Cell Biol. 89, 459–468 (2011)
15. Yang, J., Corces, V.G.: Chromatin insulators: a role in nuclear organization
and gene expression. Adv. Cancer Res. 110, 43–76 (2011)
Chapter 6
Synthesis and Posttranslational
Modifications of Proteins

6.1 Protein Synthesis

Protein synthesis (translation) is a process in which more than one hundred
macromolecules work together in a coordinated way. Translation is carried
out on ribosomes by tRNA, and on the mRNA template sequence (ch. 3.3).
Messenger RNA sequence is translated to the protein sequence. Translation
starts from the 5’ end of the mRNA and follows to 3’ end strictly in accordance
with ternary code (three nucleotides for one amino acid), that is according to
the reading frame. Even a small change in the mRNA sequence can lead to
the production of completely different protein, for example when amino acid
codon is changed to a stop codon produced protein will be shorter. Frameshift
can also occur.
A peptide chain is synthesized in the direction from amino terminus to
carboxyl terminus. It always starts from methionine, encoded by the AUG
codon (initiation codon). In bacteria, a single mRNA molecule can encode
multiple proteins (it is called a polycistronic transcript), so it contains
several initiation codons (Fig. 6.1). A protein may also contain several inner
methionines, also encoded by the AUG codon. AUG codons that serve as
initiation codons must be somehow distinguished from inner AUG codons.
This distinction occurs thanks to so called initiation signals.

6.1.1 The Initiation Signal for Protein Synthesis

The whole process of translation is very similar in prokaryotic and eukary-
otic cells. However, they have different mechanisms to recognize the initiation
codon. Many mRNA in prokaryotes are polycistronic, that means they en-
code more than one protein. Such mRNA contains a number of initiation
codons. They are recognized thanks to the specific sequence (UAAGGAGG)

W. Widłak: Molecular Biology, LNBI 8248, pp. 93–107, 2013.


c Springer-Verlag Berlin Heidelberg 2013
94 6 Synthesis and Posttranslational Modifications of Proteins

Fig. 6.1 Expression of a bacterial operon, i.e. DNA fragment encoding several
transcribed together proteins, for example structural proteins, preceded by common
control sequences — promoter and operator

located 5-10 nt upstream of initiation codon. It is called Shine-Dalgamo se-

quence (named after its discoverers). Ribosomal 16S rRNA molecule contains
a sequence complementary to the Shine-Dalgamo sequence:

Association of these two sequences leads to joining of the remaining parts of

ribosome.
Mechanism of initiation codon recognition in eukaryotes is not quite clear.
Perhaps eukaryotic ribosome simply scans the mRNA starting from the cap
at the 5’ end in search of the first AUG codon. It seems possible since almost
all eukaryotic mRNA are monocistronic (encode a single protein). However
some viral mRNA are polycistronic or do not have a cap, and are still trans-
lated in eukaryotic cell. It has been proved, that so called Kozak sequence
(named after its discoverer) — 5’-ACCAUGG — located in mRNA around
the initiation codon, increases the efficiency of translation. Also communi-
cation between the 5’ cap and the 3’ poly(A) tail of mRNA via specific
proteins (that leads to mRNA circularization) results in the enhancement of
translation.

6.1.2 Role of tRNA in Translation

Amino acids alone are unable to recognize codons in the mRNA. The amino
acid needs to be delivered to the ribosome in the form associated with the
specific tRNA — such one, which anticodon matches the amino acid codon.
6.1 Protein Synthesis 95

Molecule of tRNA with amino acid attached at the 3’ end is called the
aminoacyl-tRNA (or charged tRNA). Attachment of amino acid to tRNA
is catalyzed by different aminoacyl-tRNA synthetases (also known as activat-
ing enzymes). They recognize anticodon and the corresponding amino acid.
There are 20 different aminoacyl-tRNA synthetases present in the cell. Each
can attach only one from 20 amino acids to the corresponding tRNA. No-
tation “aa-tRNA” (amino acid-tRNA, e.g. Lys-tRNA) stands for tRNA with
attached amino acid (e.g. lysine), while notation “tRNAaa ” (e.g. tRNALys )
stands for tRNA with anticodon for the codon of given amino acid. Notation
“Lys-tRNALys ” represents the specific for lysine tRNA with lysine attached.
Once aminoacyl-tRNA enters the ribosome, complementation between its
anticodon and corresponding codon in mRNA takes place. An amino acid is
added to synthesized peptide (actually — synthesized peptide to amino acid).
In bacteria, there are 30 to 40 tRNA molecules, each with different anticodon.
In higher organisms, there are about fifty different tRNAs. What’s more
there are 61 different codons for amino acids, that is much more than tRNA.
Assuming that each codon will create pair only with completely matching
anticodon, 61 different tRNA would have to exist.

(a) (b)

Fig. 6.2 Pairing between tRNA’s anticodon and mRNA’s codon: (a) the spatial
criteria for the pairing of third base from codon are less restrictive than in the case
of other two bases; (b) possibilities of base pairing at the third codon position: for
example G can form a pair with C or U, whereas I — with C, A or U

The disproportion between the number of codons and the number of tRNA
molecules results from the fact, that there is some spatial tolerance in pair
creation of the third base in codon, which is called wobble position (Fig. 6.2a).
At this position, the normal rules for pairing are not valid: one base can create
a pair with one of several bases. Frequently, the first anticodon base (which
pairs with base at third position of the codon) is inosine (I). This allows
96 6 Synthesis and Posttranslational Modifications of Proteins

Fig. 6.3 Wobble base pairs for inosine and guanine

tRNA molecule reading of the maximum number of three codons (Fig. 6.2b
and 6.3).
The first two bases in codon form pairs with the anticodon in standard
manner — recognition is precise. More unrestricted pairing of the third base
in codon with first base of anticodon (Fig. 6.3) causes that the genetic code
is degenerated: some tRNA molecules can recognize more than one codon.

6.1.3 Process of Protein Synthesis

The whole process of translation could be divided into three stages: initiation,
elongation and termination.

6.1.3.1 Initiation
Initiation of the translation involves binding of the small subunit of ribosome
to the 5’ end of mRNA with the assistance of initiation factors (IF) (Fig.
6.4a). It is dependent on initiation signals (ch. 6.1.1). When all components
of the translational machinery are recruited, a peptide elongation starts.
Because protein synthesis always begins with methionine, the first amino-
acyl-tRNA is Met-tRNAMet i , where “i” stands for the initiation (although me-
thionine is not always the first amino acid of functional protein: N-terminus
could be cut off after synthesis). In bacteria, the methionine in initiator
aminoacyl-tRNA is modified by addition of the formyl group (-HCO) to the
amino group. This creates a formylmethionine (fMet), which is unique for
bacteria. In mammals, it induces a strong immune response.
6.1 Protein Synthesis 97

(a)

(b)

Fig. 6.4 Stages of protein synthesis: (a) without mRNA, large and small ribo-
some subunits exist separately. Protein synthesis begins when the initiation factors
(IF) form complex with the small subunit of ribosome, mRNA and the initiator
aminoacyl-tRNA. Next, the large subunit of ribosome joins, and initiation factors
are released. (b) In process of elongation one cycle comprises the following steps:
— new entry: new aminoacyl-tRNA with appropriate anticodon is bound in the
A site. In prokaryotes this phase is catalyzed by elongation factors Tu and Ts, in
eukaryotes — by EF1 and EF1β .
— peptide synthesis: the growing polypeptide connected to the tRNA in the P site
is transferred to the aminoacyl-tRNA present in A site. This process is known as
peptide bond formation. Now, the A site has the newly formed peptide, while the
P site has an uncharged tRNA (tRNA with no amino acids).
— translocation: the ribosome moves 3 nucleotides toward the 3’ end of the mRNA.
Since tRNAs are linked to mRNA by codon-anticodon base-pairing, the tRNAs
move relative to the ribosome, taking the nascent polypeptide from the A site to
the P site and moving the uncharged tRNA to the exit from the ribosome. In bacte-
ria translocation is catalyzed by elongation factor G, in eukaryotes — the elongation
factor EF2

6.1.3.2 Elongation
A ribosome contains two main tRNA binding sites: A site (aminoacyl) and
P site (peptidyl). After binding of a large subunit of ribosome with the
98 6 Synthesis and Posttranslational Modifications of Proteins

initiation complex, initiator (first) Met-tRNAMeti occupies the P site. The A

site binds the incoming aminoacyl-tRNA with the complementary codon on
the mRNA (Fig. 6.4b). Then methionine is transferred to the new aminoacyl-
tRNA and joins with its amino acid by the peptide bond. Peptidyl-tRNA is
formed, that is tRNA associated with peptide. Empty tRNA is removed from
the P site, and, at the same time, the ribosome is translocated one codon
down the mRNA chain and peptidyl-tRNA jumps from the A site to the P
site. Similar steps are repeated in subsequent cycles of elongation. The rate of
translation varies; it is up to 17-21 amino acid residues per second in proka-
ryotic cells, and up to 6-9 amino acid residues per second in eukaryotic cells.
After each cycle of elongation, the peptide is longer. The growing protein
exits the ribosome through the polypeptide exit tunnel in the large subunit.
The site by which the uncharged tRNA leaves the rybosome after it gives
its amino acid to the growing peptide chain is sometimes called the exit site
(E site).

6.1.3.3 Termination
The ribosome continues to translate codons on the mRNA until it reaches
a stop codon (UAA, UGA or UAG). These codons are not recognized by
any tRNAs. Instead, they are recognized by release factor proteins. Associa-
tion of release factor stimulates the release of peptide from ribosome. Next,
subunits of the ribosome dissociate and mRNA and tRNAs are released. All
translational components are now free for additional rounds of translation.

6.1.4 Reading Frame Shift

Reading frame shift (Fig. 6.5) is caused in most cases by insertions or deletions
of a single nucleotide in mRNA (ch. 4.4). In such situation a completely
different protein is produced.

Fig. 6.5 An example of reading frame shift. mRNA(a) and mRNA(b) differ only in
one nucleotide — it is an additional G nucleotide at the third position of mRNA(b)
(marked in green). The amino acid sequence synthesized on mRNA(b) template is
completely different starting from the insertion site.
6.2 Posttranslational Modifications and Protein Sorting 99

6.1.5 Overlapping Genes

Overlapping genes are known to be common in compact genomes (viruses,
bacteria, mitochondria). Increasing evidence suggests the existence of many
overlapping genes also in eukaryotic genomes. In the same-strand overlapping
type the shifting of reading frame enables coding for up to three completely
different proteins from one strand of DNA sequence. In eukaryotes, such type
of the gene overlapping occurs in mitochondrial DNA, where genes of, for
example, ATPase subunits 6 and 8 overlap (Fig. 6.6). ATPase 8 is composed
of 68 amino acids, ATPase 6 - of 226. Their amino acid sequence is different
even in the region encoded by a shared DNA sequence. It is interesting that
in the ATPase 6 gene there is no classical stop codon, only the first two
nucleotides (TA). Full stop codon (TAA) is formed only after the addition of
the poly(A) tail during RNA processing.

Fig. 6.6 An example of the same-strand gene overlapping

Another category of overlapping genes is based on direction of transcrip-

tion. Transcription from the same sequence in opposite directions (the dif-
ferent-strand overlapping type) leads to generation of antisense transcripts.
Their functions in living organisms are not fully understood. They could af-
fect the regulation of gene expression at the level of transcription, mRNA
processing, splicing, or translation.

6.2 Posttranslational Modifications and Protein

Sorting
A typical mammalian cell contains up to 10,000 different kinds of proteins.
For a cell to function properly, each of its numerous proteins must be local-
ized to the correct compartment. A few proteins are synthesized on ribosomes
in mitochondria and chloroplasts and are used in those organelles. However,
most proteins (even mitochondrial and chloroplast) are synthesized on ribo-
somes in the cytosol. They must be distributed to their correct destinations.
This is possible thanks to the signal sequences present in emerging peptides.
In general, segregation pathways could be divided to associated and not asso-
ciated with the endoplasmic reticulum (ER) (Fig. 6.7). Transport of proteins
to nucleus, mitochondria, chloroplasts and peroxisomes is not associated with
ER. Those proteins are synthesized on free ribosomes as soluble polypeptides.
Proteins directed to peroxisomes usually contain the sequence “Ser-Lys-Leu”
100 6 Synthesis and Posttranslational Modifications of Proteins

Fig. 6.7 General scheme of proteins sorting:1. If at the N-terminus of the new pep-
tide a fragment with hydrophobic amino acids is present, the peptide is transferred
to the endoplasmic reticulum (ER) for further sorting. If there is no hydrophobic
N-terminus, sorting is not associated with reticulum. 2. The peptide is retained in
the ER if its C-terminus contains the sequence “Lys-Asp-Glu-Leu” (KDEL in one-
letter code). If not — it is moved to the Golgi apparatus. 3. In Golgi apparatus,
proteins having a specific transmembrane helix remain. 4. After glycosylation at
the Golgi apparatus, modified proteins containing a mannose 6-phosphate (M6P)
are directed to lysosomes. 5. Proteins that aggregate with chromogranin B (secre-
togranin I) or secretogranin II are transferred to the secretory vesicles, from which
they are released upon specific stimulation (controlled secretion). Other types of
vesicles move toward the cell membrane and their content is constantly released
outside the cell (constitutive secretion).

(SKL in one-letter code) at the C terminus, while directed to mitochondria —

amphipathic helix 20-50 residues with R/K and hydrophobic sides. Transport
to the nucleus is completely different — it is described in chapter 6.2.4.
Programs available on the websites can be used to predict the protein sub-
cellular localization on the basis of amino acid sequence:
http://www.geneinfinity.org/sp/sp_proteinloc.html

6.2.1 Targeting of Proteins to the Rough Endoplasmic

Reticulum
Secretory proteins and membrane proteins are synthesized on ribosomes as-
sociated with the endoplasmic reticulum (so-called rough ER; the presence
of these bound ribosomes distinguishes the rough ER from the smooth ER).
6.2 Posttranslational Modifications and Protein Sorting 101

Fig. 6.8 Introduction of newly synthesized peptide into rough ER: (a) after synthesis
of about 70 amino acids, signal sequence present at the amino terminus of the polypep-
tide chain emerges from the ribosome. Signal sequence is recognized and bound by a
signal recognition particle (SRP). Translation is inhibited. SRP directs the peptide
with the ribosome toward the ER membrane; (b) SRP binds to its receptor on the
ER membrane. The ribosome then binds to a protein translocation complex in the
ER membrane, while SRP is released; (c) signal sequence is inserted into a membrane
channel in assistance of the signal sequence binding protein. Upon docking, polypep-
tide chain elongation resumes; (d) signal sequence is cleaved and degraded. After the
end of protein synthesis the polypeptide is released into the lumen of the ER, ribo-
somal subunits detach from the mRNA and can re-start searching for template. Re-
leased SRP also can bind another, emerging from the ribosome signal sequence.

(a) (b)

Fig. 6.9 Prevention of abnormal folding of proteins in the ER: (a) peptide may
begin folding before the end of synthesis (hydrophobic fragments have a tendency to
aggregate); (b) HSPA5 protein binds to the hydrophobic parts of emerging peptide
before they are incorrectly folded and prevents premature aggregation

Whether the ribosome will be free or attached to rough ER is determined only

by the type of protein synthesized on it. If the protein contains signal sequence
including a stretch of hydrophobic residues at the amino terminus, it will be
targeted to the ER during their translation (Fig. 6.8).
102 6 Synthesis and Posttranslational Modifications of Proteins

Inside the ER the process of protein folding takes place. Heat shock pro-
teins, dependent on the ATP, serve as chaperones which bind the growing
proteins and help in their folding (Fig. 6.9). Similar mechanisms exist also in
cytoplasm.

6.2.2 Transport of Proteins into and across the Golgi

Apparatus
Proteins without KDEL sequence at the C-terminus are directed from the en-
doplasmic reticulum to Golgi apparatus. Transport is mediated by small vesi-
cles. The Golgi is composed of stacks of membrane-bound structures known
as cisternae. An individual stack is sometimes called a dictyosome, especially
in plant cells. Each cisternae comprises a flat, membrane enclosed disc that
includes special Golgi enzymes which modify or help to modify cargo proteins
that travel through it (Fig. 6.10).

Fig. 6.10 The Golgi apparatus. Transport of proteins into and across the Golgi is
mediated by small vesicles

The main functional regions of the Golgi are: the cis-Golgi network (cis
face), medial-Golgi, endo-Golgi, and trans-Golgi network (trans face). Cis
face is directed to the endoplasmic reticulum. Vesicles from the ER fuse with
the network and subsequently progress through the stack to the trans face,
where they are packaged and sent to the required destination. Each region
6.2 Posttranslational Modifications and Protein Sorting 103

contains different enzymes which selectively modify the contents depending

on where they reside. Cis face is additionally an “emergency compartment”
for the proteins produced in the ER, which were accidentally caught in vesi-
cles flowing into Golgi apparatus (they are directed back). Vesicles released
from the trans face (maturation pole) deliver proteins and lipids to the cell
membrane, lysosomes, exosomes, endosomes, and to other destinations.

6.2.3 Transport of Proteins into Lysosomes

After glycosylation at the Golgi apparatus proteins are delivered to lysosomes
if one or more molecules of mannose are phosphorylated at the carbon atom
at position 6. Trans face of Golgi apparatus contains mannose-6-phosphate
receptor, which binds to mannose-6-phosphate present on lysosomal enzymes
and delivers them to secretory vesicles targeted to lysosomes.

6.2.4 Nuclear Transport

Many proteins produced in the cytoplasm move to the nucleus where they
perform their function. On the other hand RNA molecules produced in the
nucleus have to be transported to the cytoplasm, where there are necessary for
the protein synthesis. Macromolecules require association with karyopherins
called importins to enter the nucleus, and exportins to exit.
Small proteins with mass of 15 kDa, such as histones, can pass through
nuclear pores without the assistance of other proteins. Large proteins (above
90 kDa) can not pass through the nuclear membrane if they do not have
specific signals. Such signals may also accelerate the entry of small proteins
into the nucleus. Specific signal sequence is either directly recognized by im-
portins and exportins or through an adapter protein. For example, importin
β does not recognize a specific sequence of transported protein, but it is sup-
ported by the importin α being the adapter. On the other hand, importin
for hnRNP (heterogeneous nuclear ribonucleoprotein) recognizes a specific
sequence of hnRNP by itself. This importin is called transportin.

6.2.4.1 Nuclear Localization Signal and Nuclear Export Signal

Protein that must be imported to the nucleus from the cytoplasm contains
nuclear localization signal (NLS) that is recognized by importins. NLS is a
sequence of amino acids that acts as a tag. Two types of such signals have
been identified: SV40 type and bipartite type. SV40 type signal was found
for the first time in the large T antigen of the SV40 virus. It has amino
104 6 Synthesis and Posttranslational Modifications of Proteins

acid sequence: PKKKRKV. It is characterized by the presence of several

consecutive basic residues; in many cases it also contains a proline residue.
The bipartite type was first discovered in Xenopus neoplasmin. It contains
the sequence: KR[PAATKKAGQA]KKKK. Characteristic features are: two
basic residues, 10 spacer residues, another basic region containing at least 3-5
basic residues.
Nuclear export signal (NES) is a leucine-rich (e.g.: LQLPPLERLTL) do-
main recognized by a class of exportins called exportin 1 or Crm1. Proteins,
which are shuttled between nucleus and cytoplasm, contain both transport
signals: NLS and NES.

6.2.4.2 Mechanism of Importins and Exportins Action

Exportins and importins action is regulated by a G protein (guanine nucleotide-
binding protein) called Ran. It belongs to the family of monomeric G proteins
(known as small G proteins; there is also heterotrimeric G proteins family —
ch. 8.3). Like other G proteins, Ran may be associated with GDP (guanosine
diphosphate) or GTP (guanosine triphosphate). Switch from the GTP-bound
to the GDP-bound state is catalyzed by a GTPase-activating protein (GAP),
which hydrolyzes GTP bound to the protein. The reverse transition is cat-
alyzed by guanine nucleotide exchange factor (GEF), which induces exchange
between GDP associated with the protein and the cellular GTP (Fig. 6.11).

Fig. 6.11 Two forms of Ran: associated with GTP and associated with GDP

The GEF is located predominantly in the nucleus while GAP — almost

exclusively in the cytoplasm. Therefore, in the nucleus Ran will be mainly
loaded with GTP (Ran-GTP) while cytoplasmic Ran will be mainly in the
GDP-bound state (Ran-GDP). Binding of exportin or importin to their
cargo is dependent on the interaction with Ran: Ran-GTP enhances interac-
tions between an exportin and its cargo, and stimulates the release of cargo
from importin; Ran-GDP stimulates the release of exportin’s cargo and en-
hances the binding between an importin and its cargo. When the complex
exportin-cargo-Ran-GTP leaves the nucleus (through nuclear pores), GTP
is hydrolyzed by GAP present in the cytoplasm and the cargo is released
6.3 Protein Degradation 105

(Fig. 6.12). Similarly, importin with its cargo can move in the cytoplasm,
but the cargo is released in the nucleus, where Ran-GDP is converted to
Ran-GTP by GEF.

Fig. 6.12 General function of importins and exportins

6.3 Protein Degradation

Some proteins remain in the cell very shortly (e.g. enzymes regulating the
metabolism of the cells exist only a few minutes), others are very stable (re-
main for several days). Proteins that have fulfilled their function in the cell
or damaged proteins are targeted for degradation. Controlled protein degra-
dation is essential for the proper function of the cell. It is curried out either
in lysosomes (which contain active proteolytic enzymes) or in proteasomes.
Proteins taken in by endocytosis (derived from the outside of the cell) and
long-living proteins are mainly degraded in lysosomes. They are surrounded
by cell membrane, which then is fused with lysosomes. Short-living and dam-
aged proteins are degraded in proteasomes.
Proteasomes (26S protease complex) are large protein complexes present
both in cytoplasm and nucleus. Four stacked rings, each composed of seven
protein subunits, create core of the proteasome. At each end of the core,
regulatory units (caps) are situated. Centrally, inside the rings, catalytic cen-
ter with proteolytic properties is located, in which proteins are degraded.
Openings at the two ends of the core allow the target protein to enter.
Proteins, which are designed for destruction in the proteasome, are marked
by attachment of a few ubiquitin molecules. Ubiquitin is a small protein with
a mass 8.5 kDa. It is present in almost all tissues (ubiquitously) of eukaryotic
organisms and is conserved in evolution. A protein marked with ubiquitins
106 6 Synthesis and Posttranslational Modifications of Proteins

is digested by the proteasome while ubiquitins are released and could be

used again (Fig. 6.13). Some proteins are degraded independently of ubi-
quitination. This applies mainly to unstable proteins, which have not been
properly folded or lost their structure during a cellular stress.

Fig. 6.13 Scheme of protein degradation in the proteasome. Protein must have
attached the chain of at least 4 ubiquitin molecules in order to be recognized by
a regulatory proteasome subunit (red). Three different enzymes and energy from
ATP are necessary for the ubiquitin (Ub) attachment. Before the protein moves
into the proteasome core it must be at least partially unfolded, which also needs
the energy from ATP. After passing through the central part of the proteasome
proteins are usually degraded to 7-9 amino acids fragments.

The proteasomal degradation pathway is essential for many cellular pro-

cesses. For the discovery of ubiquitin-mediated protein degradation the 2004
Nobel Prize in Chemistry was awarded to Aaron Ciechanover, Avram Hershko
and Irwin Rose.
As a result of degradation in the proteasome generally very short peptides
are released. Sometimes however, the product of digestion in the proteasome
is functional, biologically active molecule. Some transcription factors, such as
components of NF-κB, are synthesized as an inactive precursors. Their ubiq-
uitination and degradation in the proteasome is required for transformation
into an active form. Such mechanism of selective degradation is known as
regulated ubiquitin/proteasome dependent processing (RUP).

References
1. Berg, J.M., Tymoczko, J.L., Stryer, L.: Protein Synthesis. In: Biochemistry, 5th
edn. W.H. Freeman, New York (2002)
2. Clancy, S., Brown, W.: Translation: DNA to mRNA to protein. Nature Educa-
tion 1(1) (2008), http://www.nature.com/scitable/topicpage/
translation-dna-to-mrna-to-protein-393
References 107

3. Cooper, G.M.: Protein Degradation. In: The Cell: A Molecular Approach, 2nd
edn. Sinauer Associates, Sunderland, MA (2000),
http://www.ncbi.nlm.nih.gov/books/NBK9957
4. King, M.W.: Translation of Proteins. In: themedicalbiochemistrypage.org, LLC
(1996-2012),
http://themedicalbiochemistrypage.org/protein-synthesis.php
5. King, M.W.: Protein Modifications. In: themedicalbiochemistrypage.org, LLC
(1996-2012),
http://themedicalbiochemistrypage.org/protein-modifications.php
6. Lodish, H., Berk, A., Zipursky, S.L., et al.: Protein Sorting: Organelle Biogen-
esis and Protein Secretion. In: Molecular Cell Biology, 4th edn. W.H. Freeman,
New York (2000)
7. Makalowska, I., Lin, C.F., Makalowski, W.: Overlapping genes in vertebrate
genomes. Comput. Biol. Chem. 29, 1–12 (2005)
8. Mazumder, B., Seshadri, V., Fox, P.L.: Translational control by the 3’-UTR:
the ends specify the means. Trends Biochem. Sci. 28, 91–98 (2003)
9. Muratani, M., Tansey, W.P.: How the ubiquitin-proteasome system controls
transcription. Nat. Rev. Mol. Cell. Biol. 4, 192–201 (2003)
10. Poon, I.K., Jans, D.A.: Regulation of nuclear transport: central role in devel-
opment and transformation? Traffic 6, 173–186 (2005)
11. Rape, M., Jentsch, S.: Productive RUPture: activation of transcription factors
by proteasomal processing. Biochim. Biophys. Acta 1695, 209–213 (2004)
Chapter 7
Cell Division

7.1 Cell Cycle

As the cell grows and divides, it progresses through stages in the cell cycle.
The eukaryotic cell cycle could be defined as a period from one cell division
to the next one. It consists of four phases: G1 , S, G2 and M, where “G”
stands for gap, “S” — synthesis, and “M” — mitosis. During the gaps cell in-
creases in size. After the division, cell may initiate a new round of division or
can remain for a longer period in resting phase, G0 . Cell, after appropriate
stimulation, can leave the G0 phase and re-enter the G1 phase (Fig. 7.1). In
most mammals the cell cycle lasts 12-24 hours (without G0 phase). Bacteria
divide much faster, for example E. coli — every 20-30 minutes (however,
there is no typical cell cycle in bacteria).

Fig. 7.1 Eukaryotic cell cycle phases

Proper regulation of the cell cycle is crucial to the survival of a cell. Among
others, the cell has to detect and repair damages in the DNA as well as to

W. Widłak: Molecular Biology, LNBI 8248, pp. 109–120, 2013.


c Springer-Verlag Berlin Heidelberg 2013
110 7 Cell Division

prevent uncontrolled cell division. The molecular events that control the cell
cycle are ordered and directional. Each process occurs in a sequential fashion
and it is impossible to “reverse” the cycle. The progress of the cell through the
cycle is controlled by checkpoints. They verify whether the processes at each
phase of the cell cycle have been accurately completed before progression into
the next phase. There are three major checkpoints in eukaryotic cell cycle:
– the G1 /S checkpoint (restriction checkpoint) regulates whether cells can
enter the process of DNA replication and subsequent division;
– the G2 /M checkpoint — the cell has to check a number of factors to ensure
the cell is ready for mitosis (e.g. if all DNA is correctly replicated);
– spindle checkpoint (spindle assembly checkpoint or mitotic checkpoint) —
prevents progression of mitosis until all chromosomes are properly attached
to the spindle.

7.1.1 CDK and Cyclins

Progression of the cell cycle is catalyzed by cyclin-dependent kinases (CDKs).
As the name implies, CDKs are activated by a special class of proteins called
cyclins. CDKs level remain relatively constant, while concentration of cyclins
vary in a cyclical fashion during the cell cycle. Without cyclin, CDK has little
kinase activity; only the cyclin-CDK complex is an active kinase. Different
complexes are active in different phases of the cell cycle (Fig. 7.2). CDKs
phosphorylate their substrates on serines and threonines (see Fig. 2.10c,
ch. 2.4.1) triggering specific events during cycle division such as microtubule
formation and chromatin remodeling. CDKs and cyclins are large group of
proteins. They are present in all known eukaryotes. In mammals different
CDKs are denoted by Cdk with consecutive number, and cyclins — with
consecutive (capital) letters of the alphabet.

Fig. 7.2 Cyclin-CDK complexes involved in different phases of the cell cycle in
mammals. The red line shows the stages of the cycle, in which the individual com-
plexes are present in the cell. Cell cycle begins after the creation of complex of
cyclin D (CycD) with Cdk4 or Cdk6. The final stage is catalyzed by a complex of
cyclin B (CycB) with Cdk1 (named also Cdc2). This complex is known as MPF
(maturation promoting factor or mitosis promoting factor).
7.2 Mitosis 111

7.1.2 Mechanism of Initiation and Termination of the

S Phase
In the S phase DNA replication takes place. It is triggered when all needed
proteins appear in the cell (ch. 4.1). In mammals, the expression of these pro-
teins is activated by the E2F transcription factor, which in turn is regulated
by the pRB (retinoblastoma) protein (Fig. 7.3). Binding of pRB to E2F inac-
tivates its transcriptional activity, and thus inhibits DNA replication. In late
G1 phase Cyclin D-Cdk4 complex phosphorylates pRB at the specific sites,
leading to release of E2F. E2F activates genes needed to start DNA synthesis.
It also stimulates the production of cyclin E, cyclin A and its own. Com-
plex Cyclin E-Cdk2 can also phosphorylate pRB accelerating DNA synthesis
phase. On the other hand, the complex of Cyclin A-Cdk2 can phosphory-
late E2F, inhibiting its transcriptional activity, resulting in termination of
DNA replication. It should be noted that the complex Cyclin E-Cdk2 can-
not phosphorylate E2F. If that would happen, DNA replication could end
prematurely.

Fig. 7.3 The initiation and termination of S phase

The pRB protein plays a key role in the regulation of cell cycle — it is
called “master brake of cell division”. It is a tumor suppressor protein that is
dysfunctional in many types of cancer.

7.2 Mitosis
Mitosis is the eukaryotic cell division that in conjunction with cytokinesis (cy-
toplasm division) leads to the formation of two genetically equivalent daugh-
ter cells (Fig. 7.4, Fig 1.8). There are many cells (notably among the fungi
and slime moulds) where mitosis and cytokinesis occur separately, forming
single cells with multiple nuclei.
The period between mitoses is called the interphase (includes G1 , S and
G2 phases). In the G2 phase DNA is already duplicated. The number of
chromosomes is 4n. During interphase, chromosomes are not visible in light
microscopy, because the chromatin is dispersed in the nucleus.
112 7 Cell Division

Fig. 7.4 Stages of mitosis on an example of animal cell (to simplify only two pairs
of homologous chromosomes are shown)

1. Prophase: chromatin condenses, organizing in chromosomes. Two centrioles mi-

grate to the opposite poles of the cell. From each centriole microtubules spread,
forming a structure called the mitotic spindle. At the end of prophase nuclear
membrane disintegrates into small vesicles.
2. Metaphase: chromosomes align in the middle of the cell between two centrioles.
They are guided by the microtubules attached to the kinetochore present in the
centromere of each chromosome.
3. Anaphase: Sister chromatids separate and move away from each other. Separa-
tion takes place simultaneously in all chromosomes. Crucial role in this process
is played by microtubules associated with the kinetochore.
4. Telophase: Chromosomes begin to unwind — they become less condensed. Spin-
dle disappears and nuclear membrane appears. Cell extends and finally divides
into two daughter cells (this process is called cytokinesis).

Altered from:
http://www.upt.pitt.edu/ntress/Bio1_Lab_Manual_New/mitosis_and_meiosis.htm
7.2 Mitosis 113

7.2.1 Initiation of Mitosis

Morphological changes in the early stages of mitosis are caused mainly by the
phosphorylation of group of related proteins catalyzed by mitosis promot-
ing factor (MPF — a complex of cyclin B with Cdk1). Activated MPF can
phosphorylate histones H1 leading to chromatin condensation. MPF phos-
phorylates also nuclear lamins (filament proteins of nuclear lamina), which
breaks the nuclear membrane. The structure of microtubules is dependent on
microtubule-associated proteins (MAP), which can also be phosphorylated
by MPF, what leads to the formation of the mitotic spindle. MPF activity is
controlled by phosphorylation (Fig. 7.5).

Fig. 7.5 Possibilities of MPF (CycB-Cdk1 complex) phosphorylation and its ac-
tivity. Phosphorylation can occur on tyrosine (Y) or threonine (T). MPF is active
only when it is phosphorylated at specific threonine.

7.2.2 Mechanism of Mitosis Termination

For the mitotic exit, a degradation of the mitotic cyclins is required. This is
triggered by the anaphase promoting complex (APC), a multi-subunit com-
plex which contains ubiquitin ligase activity. An inactive APC stabilizes cy-
clin A and cyclin B securing completion of DNA synthesis and progression
through G2 phase. When mitotic spindle is properly assembled, the APC is
activated by phosphorylation catalyzed by the MPF (Fig. 7.6). Mitotic cyclins

Fig. 7.6 Degradation of cyclin B leading to inactivation of MPF in negative

feedback loop
114 7 Cell Division

contain a special sequence called destruction box. It is recognized by phos-

phorylated APC that catalyzes attachment of ubiquitin molecules. Resulting
cyclins proteolysis causes the inactivation of cyclin-dependent kinases (e.g.
Cdk1, which is the part of MPF). In the absence of active MPF, lamins and
other proteins are dephosphorylated by phosphatases. This leads to restora-
tion of nuclear membrane and structures characteristic for interphase cell.

7.3 Meiosis
Meiosis is a special type of division that leads to formation of four genetically
nonequivalent daughter cells containing half the number of chromosomes of
the parental cell (that is 1n). By meiosis the germ cells, i.e. female egg cells
and male sperm, are created.
During meiosis two successive cell divisions occur (Fig. 7.7). Each division
consists of four stages similar to the stages of mitosis. However, the first
meiotic division differs from mitosis in two important aspects:

1. In the metaphase I, each pair of sister chromatids aligns together with its
homologous pair, forming two lines of sister chromatids in the equatorial
plane (in mitosis only one line is formed). This alignment is called synapsis.
At this stage crossing-over occurs, i.e. exchange of fragments of sister
chromatids between homologous chromosomes (ch. 7.4.1).
2. In the anaphase I, each pair of sister chromatids moves away from its
homologous pair, but the sister chromatids are not separated.

After the first division the number of chromosomes in each daughter cell is
2n. The second meiotic division is similar to mitosis, except that there is no
replication of DNA before the division. As a result of divisions haploid cells
are obtained (1n).

7.3.1 Independent Chromosome Segregation

In the metaphase I of meiosis, homologous pairs of duplicated chromosomes
(each consisting of two sister chromatids) line up together side by side. One
chromosome in each pair comes from the egg (the maternal homologue), the
second one comes from the sperm (paternal homologue). Homologous chromo-
somes separate into two different daughter cells. Because pairs of homologous
chromosomes are arranged randomly they are distributed to daughter cells
in a random manner. As a result, each daughter cell contains part of chromo-
somes from father and part from mother. In human cells 8.4 million (= 223 )
combinations are possible.
7.3 Meiosis 115

Fig. 7.7 Scheme of meiosis (to simplify only three pairs of homologous chromo-
somes are shown)
Altered from:
http://www.upt.pitt.edu/ntress/Bio1_Lab_Manual_New/mitosis_and_meiosis.htm
116 7 Cell Division

Fig. 7.8 Illustration of the independent segregation on example of sperm formation

from spermatocytes, which divide by meiosis. For 23 chromosomes (represented here
as circles) present in a single spermatozoon some are inherited from the mother,
the other from father. Among 8.4 million possible combinations four are shown.

7.4 DNA Recombination

DNA recombination refers to the process in which a DNA fragment is trans-
ferred from one DNA molecule to another. The most common are:
1. Homologous recombination (or DNA crossover) — occurs between two
homologous DNA molecules.
2. Site-specific recombination — occurs between two specific, identical DNA
sequences present in nonhomologous DNA.
3. Transpositional recombination — when the mobile elements are integrated
into target DNA.
Homologous recombination often occurs during meiosis or is used for
DNA repair. Other types of recombination are not specifically related to cell
division.

7.4.1 Homologous Recombination

Homologous recombination occurs between two homologous, that is iden-
tical or similar DNA molecules. It occurs during mitosis (where it is used
to repair the double-stranded breaks in DNA) and prophase I of meiosis
(pachytene), where it leads to the formation of new combinations of DNA
sequences (crossing-over). Crossing-over is a process of great importance for
genetic diversity, because it leads to the formation of daughter cells with geno-
type different than the parental cells. It consists in formation of connections
7.4 DNA Recombination 117

— chiasmata — between chromatids of neighboring homologous chromoso-

mes, and then separating those connections, so that the exchange of their
segments occurs (Fig. 7.9).

(a) (b) (c)

Fig. 7.9 Crossing-over: (a) a pair of homologous chromosomes (each containing

two sister chromatids) aligned side by side; (b) two homologous arms join together
at one point to form chiasma; (c) arms exchange DNA fragment from chiasma to
the end of chromosome

Homologous recombination is used in molecular biology as a technique for

introducing genetic changes into the organism. Thanks to the ability to per-
form homologous recombination in mammalian cells, the ability to maintain
the culture of embryonic stem cells (ES) or and obtaining chimeric animals,
in 1989 the first organism with an inactivated gene (gene knock-out) was
created. These works have been awarded with the Nobel Prize in Physiology
or Medicine in 2007 (to Oliver Smithies, Mario Capecchi and Martin Evans).

7.4.2 Site-Specific Recombination

Site-specific recombination occurs between two DNA molecules within a
short, specific DNA sequences showing only a limited degree of homology.
For the first time such recombination was observed during the integration
of phage λ DNA into the genome of E. coli. Both DNA molecules contain
sequence 5’ — TTTATAC — 3’ (called a binding site) allowing joining of two
strands of DNA according to the rules of base pairing. Next, integrase enzyme
catalyzes the formation of two single breaks (one on each DNA molecule).
After translocation of free ends, integrase cuts the other two strands and the
phage DNA is integrated into the genome.
Site-specific recombination is very specific, fast, efficient and takes place
without cofactors. It is used as a tool for genetic engineering: it enables to
118 7 Cell Division

manipulate with genetic material for testing of gene functions. In common

use is so called Cre-loxP system. The Cre (causes recombination) recombinase
from P1 phage recognizes a sequences called loxP (locus of crossing-over
P1 ). Its natural function is the separation of joined together phage genomes
created in infected E. coli. LoxP sequence, with a total length of 34 bp, con-
sists of two inverted repeats each 13 bp, and an asymmetric 8 bp core sequence
that introduces orientation to the loxP. When cells that have loxP sites in
their genome express Cre, a recombination event can occur between two loxP
sites. The result of recombination depends on the orientation of the loxP sites
(Fig. 7.10).

(a)

(b) (c) (d)

Fig. 7.10 Characteristics of Cre-loxP system: (a) the loxP structure; (b) Cre re-
combinase cuts out the DNA fragment floxed by two loxP sequences if they are
in the same orientation in the linear DNA molecule; (c) when loxP are placed in
opposite directions, the Cre recombinase leads to inversion of the floxed DNA frag-
ment; (d) when loxP are located on two different linear DNA molecules, the Cre
recombinase exchanges distal sequences. Recombination reaction can occur in both
directions (red arrow). LoxP sequences — marked with a yellow block arrow.

7.4.3 Transposition
Transposition, i.e. relocation of mobile elements from one place in the genome
to another, can proceed in two ways: by direct transfer of DNA or through
RNA (ch. 5.9). Mobile DNA elements are called transposons, while those
transferred through RNA — retrotransposons (Fig. 7.11 and 7.12). Mobile
elements are essentially molecular parasites, which appear to have no specific
function in the biology of their host organisms, but exist only to maintain
themselves. They constitute a significant portion of the genomes of many
eukaryotes.
7.4 DNA Recombination 119

Fig. 7.11 Classification of mobile elements

(a) (b)

Fig. 7.12 The mechanism of transposition: (a) transposon is cut out from the
initial DNA by a special enzyme (in bacteria it is a transposase with nuclease and
ligase activity), and then is inserted into the target DNA; (b) retrotransposons are
first transcribed from the initial DNA and the resulting RNA is “rewritten” to DNA
by reverse transcriptase and then inserted into the target DNA
120 7 Cell Division

References
1. King, M.W.: Eukaryotic Cell Cycles. In: themedicalbiochemistrypage.org, LLC
(1996-2012), http://themedicalbiochemistrypage.org/cell-cycle.php
2. Lodish, H., Berk, A., Zipursky, S.L., et al.: Regulation of the Eukaryotic Cell
Cycle. In: Molecular Cell Biology, 4th edn. W.H. Freeman, New York (2000)
3. Pray, L.: Functions and utility of Alu jumping genes. Nature Education 1(1)
(2008), http://www.nature.com/scitable/topicpage/
functions-and-utility-of-alu-jumping-genes-561
4. Pray, L.: Transposons: The jumping genes. Nature Education 1(1) (2008),
http://www.nature.com/scitable/topicpage/
transposons-the-jumping-genes-518
5. Pray, L.: Transposons, or jumping genes: Not junk DNA? Nature Educa-
tion 1(1) (2008), http://www.nature.com/scitable/topicpage/
transposons-or-jumping-genes-not-junk-dna-1211
6. Pray, L., Zhaurova, K.: Barbara McClintock and the discovery of jumping genes
(transposons). Nature Education 1(1) (2008),
http://www.nature.com/scitable/topicpage/
barbara-mcclintock-and-the-discovery-of-jumping-34083
7. Sullivan, M., Morgan, D.O.: Finishing mitosis, one step at a time. Nat. Rev.
Mol. Cell Biol. 8, 894–903 (2007)
8. Tang, Z., Hickey, I. (eds.): Cell Cycle and Cell Division. In: Miko, I. (ed.) Cell
Biology. Nature Education (2011), http://www.nature.com/scitable/topic/
cell-cycle-and-cell-division-14122649
9. Tsai, J.H., McKee, B.D.: Homologous pairing and the role of pairing centers in
meiosis. Journal of Cell Science 124, 1955–1963 (2011)
10. Wang, Y., Yau, Y.Y., Perkins-Balding, D., et al.: Recombinase technology:
applications and possibilities. Plant Cell Reports 30, 267–285 (2011)
Chapter 8
Cell Signaling Pathways

Cells have an ability to perceive and respond to their microenvironment.

External stimuli activate internal signaling pathways that regulate the cell
activity. Most signaling pathways function to transfer information from the
cell surface to effector systems.
External signals can be transmitted into the cell by several ways (Fig. 8.1):
1. As a hydrophobic molecules directly through the cell membrane;
2. Through ion channels linked receptors;
3. Through receptors associated with G protein;
4. Through receptors which are enzymes or are associated with enzymes;
5. Others (e.g. non-catalytic receptors).
In many cases, an external signal reaches the nucleus and affects gene
expression.

Fig. 8.1 Main pathways of signal transduction into the cell

8.1 Signaling through Hydrophobic Molecules

Hydrophobic molecules, such as nitric oxide (NO), arachidonic acid, or
steroids, which play an important role in intracellular signaling, can move

W. Widłak: Molecular Biology, LNBI 8248, pp. 121–138, 2013.


c Springer-Verlag Berlin Heidelberg 2013
122 8 Cell Signaling Pathways

into or from the cell directly through the cell membrane. In contrast to other
signaling cascades within a single cell, NO or arachidonic acid, formed under
the influence of external signals, can leave the cell and influence the neigh-
boring cells.
Nitric oxide is formed from L-arginine with the participation of nitric oxide
synthase (NOS). In target cells, NO stimulates cytoplasmic guanylate cyclase
to produce cyclic GMP (cGMP). Cyclic GMP (Fig. 8.2) is a secondary mes-
senger which regulates the function of many enzymes and ion channels, e.g. it
induces smooth muscle relaxation. cGMP is quite rapidly converted to GMP
by phosphodiesterase (PDE). Phosphodiesterase inhibitor is a component of
a known drug used to treat erectile dysfunction (Viagra).

Fig. 8.2 Structure of cyclic nucleotides: cAMP and cGMP, which are among the
most important secondary messengers in signaling cascades

Arachidonic acid (AA or ARA) is a polyunsaturated fatty acid that is

present in the membrane phospholipids of the body’s cells. In addition, it
is involved in cellular signaling as a lipid second messenger. It is formed
by hydrolysis of phospholipids catalyzed by phospholipase (Fig. 8.3). After
reaching the target cell it activates a protein kinase C (PKC), which phos-
phorylates some molecules in the cell altering their biological activity. Many
molecules phosphorylated by PKC are involved in e.g. processes of learning
and other processes associated with the activity of neurons. Arachidonic acid
can also be converted by other enzymes (cyclooxygenases, lipoxygenases) to
biologically active proinflammatory or antiinflammatory compounds.
Steroid hormones (glucocorticoids, mineralocorticoids, androgens, and fe-
male sex hormones; general structure shown in Fig. 1.6, in ch. 1.2.2) belong
to the superfamily of fat soluble hormones, which also includes thyroid hor-
mones, retinoids (vitamin A derivatives), and vitamin D3. Most of them enter
the cell where they bind to their receptors present in the cytoplasm or the
nucleus (only some steroid hormones bind to specific protein receptors on
the cell membrane). Steroid hormone receptor usually forms a dimeric form
after ligand (hormone in this case) binding. The main role of fat soluble hor-
mones is to regulate transcription: if ligand-receptor complex is formed in the
8.2 Signaling through Ion Channel Linked Receptors 123

Fig. 8.3 Production of arachidonic acid from phospholipid by phospholipase A2

(PLA2 )

Fig. 8.4 The mechanism of activation of transcription by steroid hormone recep-

tors. Steroid hormone receptor (SR) after joining of ligand (steroid hormone) forms
a dimer, which after binding to DNA operates together with steroid receptor coacti-
vators (SRC) and histone acetyltransferases (p300/CBP and p300/CBP-associated
factor - pCAF), what stimulates transcription. In the absence of ligand some steroid
receptors bind co-repressors (for example SMRT and NCoR) and histone deacety-
lases, which leads to inhibition of transcription.

cytoplasm, it must be transported to the nucleus, where it acts as a tran-

scription factor. It binds to specific sequences present in regulatory regions
of genes and activates or inhibits the transcription (Fig. 8.4).

8.2 Signaling through Ion Channel Linked Receptors

Ion channel linked receptors are ion-channels themselves. It is a large fam-
ily of multipass transmembrane proteins. They are involved in rapid signal-
ing events. They regulate the flow of ions across the membrane in all cells
124 8 Cell Signaling Pathways

(ch. 2.4.2.3). Hydrophilic pore (gate) inside the channel is opened or closed
depending on external factors. Ion channels may be classified by gating, i.e.
what opens and closes the channels. They are divided into three main groups:
voltage-gated ion channels, ligand-gated ion channels, and mechanosensitive
ion channels. Due to the selectivity, that is ability to pass specific types of
ions, channels are divided into cationic and anionic. When the channels are
even more “specialized” - are defined as sodium, potassium, etc. It should
be noted that the term e.g. “sodium channel” means only that sodium ions
are passed preferentialy. However, other cations could also pass through this
channel.
All cells have a resting potential: an electrical charge across the plasma
membrane, with the interior of the cell negative with respect to the exte-
rior. Sodium, potassium and chloride ions are the most important for main-
taining of the resting potential. Usually the concentration of sodium and
chloride ions outside the cell is higher than inside the cell, while the con-
centration of potassium ions is higher inside the cell. Free diffusion of ions
occurs through the cell membrane. Maintaining a constant difference of ions
concentration between the interior and exterior of cells is possible thanks to
the active (i.e. requiring energy input) transport occurring in the opposite di-
rection than diffusion. A classic example of such active transport mechanism
is the sodium-potassium pump.
The size of the resting potential varies. It can go for a long period of time
without changing significantly. However some cells (excitable cells: neurons,
muscle cells, and some secretory cells in glands), in addition to maintaining
resting potential, are able to change rapidly and transiently their membrane
potential in response to environmental or intracellular stimuli. Such short-
lasting event in which the electrical membrane potential of a cell rapidly rises
and falls, following a consistent trajectory, is called an action potential. It
is formed in excitable cell when its membrane potential exceeds a certain
limit value. Repeated generation of action potentials, that enables the flow of
sodium and potassium ions in the direction consistent with the difference in
concentrations, would lead to compensation of extracellular and intracellular
concentrations of these ions. In all excitable cells there is mechanism of active
transport, pumping ions against concentration differences and thus keeping
the concentration of ions at a constant level.
Calcium ions also play an important role in the cell. They are involved in
signal transduction pathways, where they act as a second messenger, in neu-
rotransmitter release from neurons, contraction of all muscle cell types, and
fertilization. Many enzymes require calcium ions as a cofactor. Extracellu-
lar calcium is also important for maintaining the potential difference across
excitable cell membranes, as well as proper bone formation.
The intracellular calcium level is kept relatively low with respect to the
extracellular fluid. Inside the cell, calcium ions are accumulated in the
smooth endoplasmic reticulum and mitochondria. Calcium ions can enter into
the cytoplasm either from outside the cell through the cell membrane via
8.3 Signal Transduction via Receptors Associated with G Protein 125

(a)

(b)

Fig. 8.5 Two types of calcium channels: (a) voltage-gated calcium channel located
in the cell membrane; (b) ligand-gated calcium channel located in the membrane
of endoplasmic reticulum (ER). InsP3 — triphosphoinositol.

voltage-dependent calcium channels or may be released from the endoplas-

mic reticulum by a ligand-dependent channels (Fig. 8.5).

8.3 Signal Transduction via Receptors Associated with

G Protein
Many signaling molecules bind to specific receptors on the cell membrane (the
molecule does not pass through the membrane). Such extracellular
receptors span the plasma membrane of the cell, with one part of the receptor
on the outside of the cell and the other on the inside. A ligand binding to
the outside part stimulates a transmition of the signal into the cell, elicting
a physiological response. The signal can be amplified. Thus, one signalling
molecule can cause many responses.
External signal could be transmitted via G protein-coupled receptors
(GPCRs). Although GPCRs are classically thought of working only with
G protein, they may signal through G protein-independent mechanisms
(Fig. 8.6). And also, heterotrimeric G proteins may play functional roles
independent of GPCRs.

8.3.1 G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are large family of transmembrane
proteins (in human – about 950 proteins) acting as receptors for extracellu-
lar molecules that activate intracellular signaling cascade. GPCRs mediate
a wide variety of biological processes, ranging from neurotransmission and
126 8 Cell Signaling Pathways

Fig. 8.6 Scheme of signal transduction in cell through the receptors associated
with G protein. Activated G protein transfers the signal to one of the effectors
what leads to production of the appropriate second messengers.

hormonal control of virtually all physiological responses, to perception of

taste, smell, light, and pain. Such receptors are found only in eukaryotes and
can bind with a number of ligands listed in the diagram shown in Fig. 8.6 and
in ch. 2.4.2.2 (each ligand recognizes its own receptor). Substance (ligand)
that binds to the receptor and triggers a response by that cell is called an
agonist. Whereas an agonist causes an action, an antagonist binds to the
receptor and blocks it preventing the activation by agonist.

8.3.2 G Proteins
Binding of a ligand to the GPCR causes a conformational change in the
receptor. This leads to activation of G protein associated with the receptor,
by exchanging its bound GDP for a GTP (Fig. 8.7). G proteins (guanine
nucleotide-binding proteins) associated with the GPCRs belong to the family
8.3 Signal Transduction via Receptors Associated with G Protein 127

Fig. 8.7 Changes of G protein between inactive and active form: (a) in the inactive
form, α subunit is associated with GDP (guanosine diphosphate); (b) interactions
between the receptor activated by the agonist and the α subunit lead to the release
of GDP. GTP binds in the empty site (its concentration in the cell is much higher
than the concentration of GDP); (c) α subunit associated with GTP has small
affinity to the βγ complex, which leads to separation of subunits, so they can
activate further signal molecules; (d) α subunit possess GTP-ase activity: it finally
hydrolyzes GTP to GDP, what leads to inactivation of the G protein.

(a) (b)

(c)

Fig. 8.8 Transmission of signal from the receptor associated with G protein to
effector: (a) before the agonist binds to the receptor, three subunits of the G pro-
tein form a complex; (b) after binding of agonist, the receptor affecs G protein,
changing the GDP in the α subunit for the GTP, so the subunit becomes active;
(c) α subunit and γβ complex are separated and can interact with effector pro-
teins (this figure shows the interaction with adenylyl cyclase responsible for cAMP
production). Both, α subunit and γβ complex, can interact with different effectors
(listed in Fig. 8.6).
128 8 Cell Signaling Pathways

of heterotrimeric G proteins (composed of α subunit, and β and γ subunits

forming the stable complex). They are associated with the inside surface of
the cell membrane and serve as a “dashboard” on the way between receptor
and effector (Fig. 8.6 and 8.8). G proteins are highly differentiated: over a
dozen genes encoding α subunit have been cloned so far (Table 8.1). Also β
and γ subunits may exist in different variants. They behave differently in the
recognition of the effector, but share a similar mechanism of activation.

Table 8.1 Mammalian G protein α subunits and their effectors. Intracellular com-
munication via G proteins could be affected by bacterial toxins. After binding of
the cholera toxin, GTP associated with Gα can not be hydrolyzed to GDP and
the G protein remains all the time in the active form. By contrast, pertussis toxin
prevents GDP release from the α subunit and G protein is blocked in the inactive
state.
Class Gene variant Effectors Sensitivity to toxins
Gαs αs(s) (+) adenylate cyclase
αs(L) (+) calcium channel cholera
(-) sodium channel
αolf (+) adenylate cyclase cholera
Gαi/o αi1 (-) adenylate cyclase
αi2 (-) calcium channel pertussis
αi3 (+) sodium channel
αoa (-) calcium channel
αob (+) phospholipase C pertussis
(+) phospholipase A2
αt−r cholera
αt−c (+) cGMP phosphodiesterase and pertussis
αg (+) phospholipase C pertussis
αz (-) adenylate cyclase
Gαq/11 αq
α11 (+) phospholipase C
α14
α15/16
Gα12/13 α12
α13 (+) small G protein
Notes: (+) activation; (-) inhibition

G proteins were discovered by Alfred G. Gilman and Martin Rodbell when

they investigated stimulation of cells by adrenaline. For this discovery, they
won the 1994 Nobel Prize in Physiology or Medicine.

8.3.3 Effectors and Secondary Messengers

Both activated α subunit and released βγ subunits of the G protein can acti-
vate different signaling cascades and effector proteins (listed in the diagram
8.3 Signal Transduction via Receptors Associated with G Protein 129

in Fig. 8.6). The main effectors on which α subunit acts are listed in table 8.1
(γβ complex also can act on the same effectors). Adenylate cyclase, a mem-
brane protein, catalyzes the conversion of ATP to cyclic AMP, one of the
most important secondary messengers (Fig. 8.2; ch. 8.5.1). Whereas guany-
late cyclases (membrane-bound or soluble forms) catalyze the formation of
cGMP. Phospholipase A2 (PLA2 ) cleaves phospholipids generating the for-
mation of arachidonic acid (Fig. 8.3). Phospholipase C (PLC) also cleaves
phospholipids, but in a different position than PLA2 , leading to the forma-
tion of diacylglycerol (DAG) (Fig. 8.9). Among the variety of PLC (β, γ, δ,
and others), PLC-β is activated by G protein.

Fig. 8.9 An example of the reaction catalyzed by phospholipase C (PLC): hy-

drolysis of phosphatidylinositol diphosphate generates the formation of secondary
transmitters: diacylglycerol (DAG) and triphosphoinositol (InsP3 , IP3 ). DAG re-
mains in the membrane and activates protein kinase C (PKC)

Second messengers produced by effectors relay signals to target molecules

inside the cell, in the cytoplasm or nucleus. They greatly amplify the strength
of the signal.
There are three basic types of secondary messenger molecules:
– hydrophilic (i.e. water-soluble), like cAMP, cGMP, InsP3 , and calcium ions,
that are located within the cytosol;
– hydrophobic (i.e. water-insoluble), like diacylglycerol, and phosphatidyli-
nositols, which are membrane-associated and diffuse from the plasma mem-
brane into the intermembrane space where they can reach and regulate
membrane-associated effector proteins;
– gases: nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide
(H2 S) which can diffuse both through cytosol, and across cellular
membranes.
For discovery of second messengers, Earl Wilbur Sutherland, Jr. won the 1971
Nobel Prize in Physiology or Medicine.
130 8 Cell Signaling Pathways

8.4 Signal Transduction through Enzyme Linked

Receptors
Enzyme-linked receptors are either enzymes themselves, or are directly as-
sociated with the enzymes that they activate. These are usually single-pass
transmembrane receptors, with the enzymatic portion of the receptor being
intracellular. The majority of enzyme-linked receptors are protein kinases, or
associate with protein kinases, mainly tyrosine or serine/threonine kinases.
These kinases phosphorylate respectively tyrosine and serine or threonine in
target proteins. They are capable of autophosphorylation as well as phos-
phorylation of other substrates. Protein phosphorylation leads to changes in
its enzymatic activity, intracellular localization, or in interactions with other
proteins. Protein kinases are also found in the cytosol in the form of enzymes
not associated with the receptors.
Abnormal activity of receptor kinases is a common cause of diseases, espe-
cially carcinogenesis. Signaling pathways affected by such kinases could serve
as targets for cancer therapy. Some drugs that are inhibitors of protein ki-
nases are already approved for treatment, other drugs are in clinical trials
stage.

8.4.1 Receptor Tyrosine Kinases

Of the 91 unique tyrosine kinases identified so far, 59 are receptor tyrosine
kinases (RTKs). They are transmembrane proteins: the domain with tyrosine
kinase activity is located on the cytoplasmic side (together with regulatory
domain). Extracellular region contains ligand binding domain. It can be a sep-
arate unit connected with the rest of the receptor by disulfide bond. Receptor
tyrosine kinases are involved in transmitting signals into the cell, and their ac-
tivity is extremely important in the regulation of cell division, differentiation,
and morphogenesis. The majority of them are receptors for growth factors
and hormones like epidermal growth factor (EGF), platelet derived growth
factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor
(HGF), insulin, nerve growth factor (NGF), vascular endothelial growth
factor (VEGF), macrophage colony-stimulating factor (M-CSF), etc.
Binding of ligand to the receptor leads to oligomerization (usually dimer-
ization) of monomeric receptor kinase or stabilization of already existing
loosely associated dimers. Thereafter trans-autophosphorylation of kinases
(i.e. phosphorylation of neighboring kinase in dimer) occurs. As a result of
phosphorylation, changes in the spatial structure of the receptor are gener-
ated, causing the opening of kinase domain and binding of ATP in catalytic
center (Fig. 8.10). Activated tyrosine kinase phosphorylates specific target
proteins - often they are also enzymes, such as cytoplasmic tyrosine kinases.
8.4 Signal Transduction through Enzyme Linked Receptors 131

This starts the kinases cascade, in which further types of kinases are phos-
phorylated and activated. As a result, transcription factors coud be activated
or inactivated leading to changes in the expression of specific genes.

(a) (b) (c)

Fig. 8.10 General model of regulation of the activity of receptor tyrosine kinases:
(a) in the absence of ligand the tyrosine kinase domain (dark green) of a recep-
tor maintains the basal, low activity due to inhibitory interactions with the region
adjacent to the membrane (red) and/or the carboxyterminal tail (violet). In addi-
tion, the activation segment (brown) has a structure that blocks an access to the
catalytic center; (b) after binding of ligand (orange) and dimerization of the extra-
cellular domains (yellow), the cytoplasmic domains are juxtaposed, which facilitates
the trans-autophosphorylation of tyrosine residues (shown as circles) in the region
adjacent to the membrane, in the activation segment and in the carboxy-terminal
tail; (c) after phosphorylation (black dots) and reconfiguration of the inhibitory seg-
ments, the kinase domains become fully active (light green). Then phosphotyrosines
interact with proteins containing SH2 (Src Homology 2) domains or phosphotyro-
sine binding domains. This initiates a series of events which eventually result in
altered patterns of gene expression or other cellular responses.
Altered from: http://www.nature.com/nrm/journal/v5/n6/fig_tab/nrm1399_F1.html
Hubbard SR (2004) Juxtamembrane autoinhibition in receptor tyrosine kinases.
Nature Reviews Molecular Cell Biology 5, 464-471.

Many ligands of receptor tyrosine kinases are multivalent. Also some ty-
rosine receptor kinases may form heterodimers with similar but not identical
kinases. It is the reason why the response to the extracellular signal can be
highly varied.
Cell must have a possibility to stop the signal response. Constant activity
of receptors, e.g. growth factors, would lead to the uncontrolled division and
consequently to carcinogenesis. Ligand-receptor complex, in case of receptor
tyrosine kinases, is absorbed and destroyed through receptor endocytosis.
A class of tyrosine kinases recruited to the receptor just after connection
of ligand to it is involved in signaling cascades activated among others by cy-
tokines. One of such kinases is JAK kinase (Janus kinase) regulating activity
of STAT proteins (ch. 8.5.3). Cytokines are protein molecules that affect
132 8 Cell Signaling Pathways

growth, proliferation and stimulation of hematopoietic cells and cells involved

in immune response.

8.4.2 Receptor Serine/Threonine Kinases

At least 125 of more than 500 human protein kinases are serine-threonine
kinases (STKs). Their activity is regulated by cAMP/cGMP, diacylglycerol
(DAG), Ca2+ /calmodulin, or DNA damage. STKs select specific residues to
phosphorylate on the basis of residues that flank the phosphoacceptor site,
which together comprise the consensus sequence.
Receptor serine/threonine kinases transmit mainly signals induced by
growth factors belonging to the family of TGFβ (transforming growth fac-
tor β). They exist as heterodimers consisting of type I and type II receptor.
Ligand binding domain is present in type II receptor. Type II receptor binds
ligand and then recruits and phosphorylates type I receptor. Activated type
I receptor phosphorylates specifically SMAD proteins, which are transported
to the nucleus where regulate gene expression.

8.5 Intracellular Signaling Pathways

There are a large number of intracellular signaling pathways responsible for
transmitting information within the cell. The majority respond to external
stimuli, which are received by receptors embedded in the plasma membrane.
These receptors then transfer information across the membrane using a va-
riety of transducers and amplifiers that engage a diverse repertoire of intra-
cellular signaling pathways. The other categories are the pathways that are
activated by signals generated from within the cell. All of these signaling
pathways generate an internal messenger that is responsible for relaying in-
formation to the sensors that then engage the effectors that activate cellular
responses. Signaling pathways often interact with each other. Interactions
(positive and negative) of second messengers systems (so called cross-talk)
decide on the final activity of different pathways and biological response.
Signaling pathways are dynamic and can proceed differently in different cell
types. Several examples of the better known cell signaling pathways are out-
lined below.

8.5.1 cAMP-dependent Signaling Pathway

Cyclic AMP is involved in the activation of protein kinases and regulates the
effects of adrenaline and glucagon. It also binds to and regulates the function
of ion channels dependent on cyclic nucleotides and other factors. Protein ki-
nase A (PKA) is the main target of cAMP. PKA is present in the cytoplasm
in an inactive form as tetramer (R2C2), composed of two catalytic subunits
(C) and two regulatory subunits (R). Usually, it is located in the perinuclear
8.5 Intracellular Signaling Pathways 133

space (thanks to the AKAP protein - membrane-associated archoring pro-

tein). Cyclic AMP binds to regulatory subunits, which leads to the release
of PKA catalytic subunits. Active PKA can phosphorylate among others:
enzymes associated with the synthesis of neurotransmitters and metabolism
of cyclic nucleotides, neurotransmitter receptors, ion channel proteins, pro-
teins involved in regulation of transcription and translation, or cytoskeletal
proteins. Processes dependent on cAMP/PKA (as well as cGMP/PKG) are
mostly short-term processes, because rapid dephosphorylation of these pro-
teins occurs. A more prolonged cellular responses are associated with the
influence of cAMP on gene expression (Fig. 8.11). Released PKA catalytic
subunits migrate to the nucleus (by passive diffusion), where they phospho-
rylate CREB protein (cAMP response element-binding protein). This leads
to transcription of genes containing CRE sequence (cAMP response element)
in the promoters (ch. 5.3.1).

Fig. 8.11 Example of the complexity of processes dependent on cAMP/PKA.

cAMP binds to two sites in each of the regulatory subunits (R) of PKA (protein
kinase dependent on cAMP). Released catalytic subunits (C) phosphorylate serines
and threonines in the target proteins. Long-term PKA activity is associated with
effects on the gene expression through CREB. CREB activity can also be regulated
by other enzymes: calmodulin, nerve growth factor NGF, and phosphatases. PKA
phosphorylates at the same time CREB, I-1 protein and NIPP-1 protein. I-1 in
phosphorylated form is an inhibitor of protein phosphatase PP1, while NIPP-1 in
dephosphorylated form is an inhibitor of PP1, thus in phosphorylated form has
activity opposite to the phosphorylated I-1. PP1 activity is a function of activities
of phosphorylated proteins I-1 and NIPP-1. If PP1 is active, it dephosphorylates
proteins phosphorylated by PKA.

8.5.2 NF-kappaB Signaling Pathway

NF-κB transcription factor can be activated by many signals, including TNF-
α (tumor necrosis factor α), interleukin 1 (IL-1), compounds triggering mi-
tosis (mitogens) in T and B cells, bacterial lipopolysaccharides (LPS), viral
134 8 Cell Signaling Pathways

proteins, and others. It controls the expression of genes involved in immune

response, apoptosis, and cell cycle regulation. Therefore, the abnormal regu-
lation of NF-κB may cause an inflammation, autoimmune diseases, viral in-
fections, and carcinogenesis. In mammals, the family of NF-κB includes five
proteins: NF-κB1 (or p50), NF-κB2 (or p52), RelA (or p65), RelB, and c-
Rel. All of them possess conserved in evolution Rel domain, responsible for
dimerization, binding to DNA, and for binding of IκB (inhibitor of NF-κB).
NF-κB works only as a dimer. The most common active form of NF-κB
contains subunits p50 or p52, and p65.
In the cytoplasm, NF-κB is inhibited by IκB. Activation signal transmitted
from receptor (e.g. after binding of TNF-α to its receptor) leads to phospho-
rylation of the inhibitor by the IKK kinase (IκB kinase). This results in
ubiquitination and degradation of IκB in the proteasome. Released NF-κB
migrates to the nucleus, where it activates transcription of various genes, in-
cluding its inhibitor, which within one hour reappears in the cell (Fig. 8.12).

Fig. 8.12 General model of NF-κB activation

8.5.3 JAK-STAT Signaling Pathway

The JAK-STAT system consists of three main components: a receptor, JAK,
and STAT. The receptors do not possess catalytic kinase activity, thus they
8.5 Intracellular Signaling Pathways 135

rely on the JAK family of tyrosine kinases. JAK is short for Janus Kinase.
STAT proteins (Signal Transducer and Activator of Transcription) (family
of seven proteins) are proteins both transmitting signal and activating tran-
scription. The inactive form is a monomer and is in constant motion between
the cytoplasm and the nucleus awaiting for activating signal. The JAK-STAT
system is a major signaling alternative to the second messenger system.
Signal activating the JAK-STAT pathway comes from the membrane re-
ceptors. They can be activated by interferon, interleukin, growth factors, or
other chemical messengers. The receptors exist as paired polypeptides and
JAKs are associated with intracellular domains. Ligand binding enables a
conformational change of the receptor and then JAK kinase autophosphory-
lates itself. The STAT protein then binds to the receptor. STAT is phosphory-
lated, forms a dimer (through the SH2 domain) and translocates into the cell
nucleus, where it binds to DNA and promotes transcription of genes respon-
sive to STAT (Fig. 8.13). STAT protein is dephosphorylated and inactivated
by nuclear phosphatases, and then transported to the cytoplasm.

(a) (b) (c) (d)

Fig. 8.13 General model of STAT protein activation

8.5.4 MAPK Signaling Pathway

Mitogen-Activated Protein Kinases (MAPKs) are serine/threonine-specific
protein kinases. They respond to variety of external signals (mitogens, os-
motic stress, heat shock, and proinflammatory cytokines), resulting in either
cell growth, differentiation, inflammation, or apoptosis. Generally, the signal
is transmitted as follows (“MAPK cascade”):
stimulus → MAPKKK → MAPKK → MAPK → response,
where MAPKK is MAPK kinase, and MAPKKK - MAPKK kinase.
136 8 Cell Signaling Pathways

Ligands that activate MAPK’s cascade bind receptor tyrosine kinases. Next,
signal is transmitted by small monomeric G proteins (sometimes by calcium
ions or certain heterotrimeric G proteins) to MAPKKK. MAPK regulates
the activities of several transcription factors (e.g. MYC, FOS), what leads to
altered transcription of genes that are important for the cell cycle. Another
effect of MAPK activation is to alter the translation of mRNA to proteins.

8.6 Apoptosis
Apoptosis, also known as programmed cell death (PCD), is strictly regu-
lated, active process of self-destruction of an individual cell that may occur
in multicellular organisms. It is an important cellular process that starts by
specific signaling pathways in response to some external and internal stim-
uli. Morphologically, apoptosis is characterized by chromatin condensation
and shrinkage of cell, and then by fragmentation of the nucleus and cyto-
plasm. As a result, apoptotic bodies surrounded by the cell membrane are
formed, which are absorbed by phagocytes. In contrast to apoptosis, cells that
die by necrosis swell and disintegrate, releasing the contents, which induces
inflammation.
Apoptosis is an important process in development of organisms. Redun-
dant cells are removed by apoptosis (e.g. in human embryo, during digit
formation cells between fingers are removed and lack of apoptosis can lead to
webbed fingers called syndactyly). Apoptosis is also important in the aging
process and in many diseases. Excessive apoptosis occurs in many autoim-
mune and autodegenerative diseases, while not sufficiently efficient apoptosis
is often one of the causes of the carcinogenesis.
A characteristic molecular feature of cells undergoing apoptosis is activa-
tion of specific cysteine proteases called caspases. Until now more than 10
different caspases have been identified. Caspases are involved in the signaling
cascade, in which consecutive enzymes are activated as a result of limited
proteolysis. Some of them, called initiator (apical, e.g. caspase-2, -8, -9, and
-10), are activated by specific stimulus and start a cascade. Others, called
effector (executioner, e.g. caspase-3, -6, and -7), are activated by initiator
caspases and catalyze the proteolysis of other proteins essential for cellular
functions. Signaling pathway associated with caspases includes:

1. Activation of apical caspases by specific initiatory signals, e.g. changes in

membrane receptors status.
2. Activation of effector caspases by initiatory caspases, which cut inactive
caspases (procaspases) at specific sites.
3. Specific proteolysis of important cellular proteins by effector caspases. One
of the substrate for effector caspases is inhibitor (DFF45/ICAD protein)
of apoptotic nuclease (DFF40/CAD). The cleavage and inactivation of
8.6 Apoptosis 137

the inhibitor allows nuclease to enter the nucleus and fragment the DNA,
which is an irreversible stage of apoptotic cell death.
Signals inducing apoptosis can reach the cell from the outside. Death re-
ceptor (extrinsic) pathway of apoptosis is then triggered (Fig. 8.14). So called
death ligands (e.g. FasL/CD95L, TRAIL, APO-3L, or TNF) bind with cell
membrane specific death receptors (Fas/CD95, DR3, DR4, DR5, TNFR) pos-
sessing the death domain (DD). Binding of ligand to receptor induces its
trimerization and attachment of adaptor proteins, also containing death do-
mains (FADD, TRADD, RAIDD, DAXX, and others). As a result, initiatory
caspases-8 or -10 are activated.

Fig. 8.14 Simplified diagram of transmitting signals leading to apoptosis

Apoptosis can also be caused by intracellular factors, mainly by signals

from the mitochondria (mitochondrial or intrinsic pathway of apoptosis).
Factors leading to increase of mitochondrial membrane permeability may in-
fluence release of proapoptotic factors (such as cytochrome c) into the cytosol.
Released cytochrome c, together with APAF1 protein activates the initiator
caspase-9, which then activates executioner caspase-3.
In cells undergoing apoptosis the process of cytochrome c (and other apop-
totic proteins) release from mitochondria is regulated by a number of sup-
porting proteins from Bcl-2 family. This family consists of three subfamilies.
Two of them are formed by proteins present in the outer mitochondrial mem-
brane: pro-apoptotic Bax and Bak proteins, and anti-apoptotic Bcl-2 and
138 8 Cell Signaling Pathways

Bcl-xL proteins. These proteins in mitochondrial membrane can form homo-

or heterodimers. The third subfamily, including Bid, Bik, and Bim, includes
pro-apoptotic proteins present in the cytoplasm. They can interact with mem-
brane proteins from Bcl-2 family and modify their activity. It is believed that
membrane proteins from Bcl-2 family can form membrane pores enabling
outflow of cytochrome c to the cytoplasm or can interact with proteins form-
ing ion channel VDAC (voltage-dependent anion channel) and modulate its
permeability to cytochrome c.

References
1. Balla, T., Szentpetery, Z., Kim, Y.J.: Phosphoinositide signaling: new tools and
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2. Chen, R.E., Thorner, J.: Function and regulation in MAPK signaling pathways:
lessons learned from the yeast Saccharomyces cerevisiae. Biochim. Biophys.
Acta 1773, 1311–1340 (2007)
3. Gewies, A.: ApoReview - Introduction to Apoptosis (2003),
http://www.celldeath.de/encyclo/aporev/aporev.htm#Index
4. Hayden, M.S., Ghosh, S.: Signaling to NF-kappaB. Genes Dev. 18, 2195–2224
(2004)
5. Hille, B.: G protein-coupled receptor. Scholarpedia 4(12), 8214 (2009),
http://www.scholarpedia.org/article/G_protein-coupled_receptor
6. Hille, B.: Ion channels. Scholarpedia 3(10), 6051 (2008),
http://www.scholarpedia.org/article/Ion_channels
7. Hubbard, S.R.: Juxtamembrane autoinhibition in receptor tyrosine kinases. Na-
ture Review Molecular Cell Biology 5, 464–471 (2004)
8. King, M.W.: Signal Transduction. In: themedicalbiochemistrypage.org, LLC
(1996-2012),
http://themedicalbiochemistrypage.org/signal-transduction.php
9. Kroeze, W.K., Sheffler, D.J., Roth, B.L.: G-protein-coupled receptors at a
glance. Journal of Cell Science 116, 4867–4869 (2003)
10. Lodish, H., Berk, A., Zipursky, S.L., et al.: Cell-to-Cell Signaling: Hormones
and Receptors. In: Molecular Cell Biology, 4th edn. W.H. Freeman, New York
(2000)
11. Neitzel, J., Rasband, M. (eds.): Cell Communication. In: Miko, I. (ed.) Cell
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12. Rawlings, J.S., Rosler, K.M., Harrison, D.A.: The JAK/STAT signaling path-
way. Journal of Cell Science 117, 1281–1283 (2004)
Chapter 9
High-Throughput Technologies in
Molecular Biology

Over the past few decades, advances in the computer science and informatics
have made possible development of new strategies in molecular biology that
enable to collect and analyze the data about complex interactions within bio-
logical systems. A new inter-disciplinary field of study, called systems biology,
has been created. It analyzes and integrates information from genomics (DNA
level), transcriptomics (RNA level), proteomics (protein level) and other re-
lated fields (e.g. metabolomics and other -omics). Specialized tools to view
and analyze biological data and computerized databases to store, organize,
and index the data are required. Thus, bioinformatics, the interface between
the biological and computational sciences, is indispensable to obtain a clearer
insight into the fundamental biology of organisms.

9.1 DNA Sequencing

DNA sequencing is the laboratory method used to determine the exact order
of base pairs in a DNA fragment. The DNA sequence contains information
necessary for organisms to survive and reproduce, thus reading the sequence
is useful in fundamental research into why and how organisms live. With the
sequencing of genomes, genomics was developed, which can be defined as the
mapping of genes of various organisms by large-scale DNA-sequence analysis.

9.1.1 Chain Termination Sequencing

The first techniques of sequencing were established in the 1970-1980s. One
of the major methods is known as chain termination sequencing, dideoxy
sequencing, or Sanger sequencing after its inventor Frederick Sanger (Nobel
Prize in Chemistry in 1980, shared with Walter Gilbert). The single stranded
DNA fragment (which is the sequence to be determined) serves as a template

W. Widłak: Molecular Biology, LNBI 8248, pp. 139–153, 2013.


c Springer-Verlag Berlin Heidelberg 2013
140 9 High-Throughput Technologies in Molecular Biology

Fig. 9.1 Chain termination method of DNA sequencing. The synthesis of a new
chain stops after incorporation of dideoxynucleotide (ddNTP). If ddNTPs are la-
beled differently, products of the reaction can be analyzed together, if not - samples
with each ddNTP should be analyzed separately.
9.1 DNA Sequencing 141

for DNA polymerase, that normally uses deoxynucleotides (dNTPs) to syn-

thesize a new strand starting from the primer complementary to the given
sequence (ch. 4.1.1). In the Sanger method, dideoxynucleotides (ddNTPs) -
nucleotides missing a hydroxyl (-OH) group at the 3’ position (see Fig. 3.2),
are added to the reaction. To this position normally a new nucleotide is at-
tached in condensation reaction (Fig. 3.5). If there is no -OH group in the 3’
position, the additional nucleotides cannot be added to the chain, thus inter-
rupting chain elongation. In the manual method of sequencing one of dNTPs
or primer are labeled (e.g. by radioactive sulfur) and the sample has to be di-
vided into four tubes, each containing all dNTPs and a smaller amount of one
of ddNTPs (ddATP, ddTTP, ddCTP or ddGTP). During reaction ddNTPs
have a chance to be incorporated in each position in the sequence. Provid-
ing that there is enough substrates in the reaction tube, both template and
nucleotides, the obtained terminated products will be of all possible lengths,
differing in size by one nucleotide. Finally, the mixture of varying length
fragments is run on a gel and the sequence can be read from the smallest
fragment to the largest (Fig. 9.1).
Since 1986 the process of sequence reading can be done with an automated
fluorescence sequencer. Automated sequencing uses fluorescent tags on the
ddNTPs (a different dye for each nucleotide). This makes it possible for all
four reactions to be run in one lane and increases the speed of the process.
A laser constantly scans the bottom of the gel, detecting the bands that
move down the gel. The runs are fully automated nowadays, and the gels are
replaced by capillaries.

9.1.2 Full Genome Sequencing

Chain termination methods (sometimes referred to as first generation se-
quencing) can directly sequence only relatively short (300 - 1,000 nucleotides
long) DNA fragments in a single reaction. Thus, longer sequences (like ge-
nomic DNA) has to be fragmented (with restriction enzymes or mechanical
forces) into random pieces. In conventional genome sequencing, DNA frag-
ments are subcloned into vectors (e.g. plasmids) and introduced into bacterial
cells to prepare a library covering the whole genome. The transformed cells
containing subclones are plated and grown and then the DNA from each one
is isolated and sequenced in a capillary sequencer (Fig. 9.2a). The sequence
is assembled electronically into one long, contiguous sequence by using over-
lapping DNA regions. Such method of genome sequencing (called shotgun
sequencing technology) was applied to sequence the first human genome. Se-
quencing of the essentially complete genome (by The Human Genome Project
and Celera Genomics) took 13 years and was announced in April 2003. The
sequence of the DNA along with sequences of known and hypothetical genes
142 9 High-Throughput Technologies in Molecular Biology

(a)

(b)

Fig. 9.2 Comparison of conventional chain termination sequencing (a) and mas-
sively parallel sequencing (b). Altered from: Mardis E.R. (2006) Anticipating the
1,000 dollar genome. Genome Biol. 7, 112.

and proteins is stored in databases such as GenBank housed by the National

Center for Biotechnology Information.
In another method for in vitro clonal amplification, genomic DNA frag-
ments first undergo end-repair to provide blunt ends for adaptor ligation.
Next, they have specific adaptors ligated to their ends that contain priming
sites for PCR (polymerase chain reaction, see ch. 4.1.2) and sequencing. The
adaptor-ligated fragments are then hybridized to complementary adaptors
that are fixed to a surface (a slide or bead). Then in situ PCR amplification
is used instead of bacterial amplification. Sequencing reactions of the ampli-
fied fragments take place on the surface (Fig. 9.2b). The sequence is visualized
base by base simultaneously in all fragments using either luciferase or fluores-
cence reporting that is detected by a CCD camera. Such technology, named
massively parallel genome sequencing (or second generation sequencing), was
announced in 2005. It has reduced the cost and increased the throughput
of genomic sequencing. Nowadays smaller genomes can be synthesized in one
experiment. The technology is widely used also for microRNA screening, gene
expression analyzes and ChIP-seq (see ch. 9. 3).
The second generation technologies were initially inferior to classical se-
quencing in terms of read length (about 35-76 nt for Illumina platform versus
about 700 nt for classical sequencing) and single-read error rate (about 1-
2% versus less than 0.1%). Rapidly developing third generation technologies
9.2 Analysis of Gene Expression at RNA Level 143

(frequently identified with single-molecule sequencing) provide solutions to

some of the problems that second generation sequencing faced by simplify-
ing sample preparation, reducing sample mass requirements, and eliminating
amplification of DNA templates. There are several different strategies of fast
sequencing and a number of public and private companies are competing
to commercialize full human genome sequencing. However, we should keep in
mind that although full genome sequencing provides raw data on all six billion
letters in an individual’s DNA, it does not provide an analysis of what that
data means or how that data can be utilized in various clinical applications,
such as in medicine to help prevent disease.

9.2 Analysis of Gene Expression at RNA Level

The expression of genes can be determined by measuring mRNA levels. For
a single gene, Northern blotting can be used. The technique is based on
nucleic acids hybridization, that is preferential binding of complementary
single-stranded nucleic acid sequences (see ch. 3.2.2). The total RNA is iso-
lated from cells and after separation in a gel (electrophoresis) bound to the
membrane. A labeled probe (introduced into the solution in single-stranded
form) specific for the examined gene can hybridize and leave the signal only
with RNA molecules with a complementary sequence. Nowadays, the most
useful method to evaluate the expression of a single gene is the RT-PCR
(reverse transcription - polymerase chain reaction) technique (especially real
time RT-PCR, which is a quantitative method). Reverse transcriptase first
copies RNA to DNA (such DNA created from RNA is called cDNA - comple-
mentary DNA), next the examined sequence is amplified in a series of PCR
cycles by DNA polymerase using specific primers (ch. 4.1.2; Fig. 4.4).
The complete genome sequencing has opened the way to the investigation
of the transcriptional activity of thousands of genes (i.e. the transcriptome)
in selected tissues, in one experiment through DNA microarrays. Microarrays
are producing massive amounts of data. These data, like genome sequence
data, can help us to gain insights into underlying biological processes. The
structural analysis of accumulated transcripts in a cell or tissue is the main
goal of transcriptomics.

9.2.1 DNA Microarrays

The basic use of microarrays (arrays, chips) is gene expression profiling, thus
RNA detection. Microarrays can be used also to detect DNA: in studies
of DNA-protein interactions (ch. 9.3), in comparative genomic hybridization,
144 9 High-Throughput Technologies in Molecular Biology

chromatin modifications, SNPs (single nucleotide polymorphisms) detection,

and in other applications. Microarrays studies, similarly to Northern blotting,
are based on nucleic acids hybridization. The main difference between these
two techniques is that in Northern blotting RNA is fixed with a surface and
one labeled probe is used in a hybridization solution, while in microarrays
multiple probes are attached at fixed locations (spots) to a surface (typically
a glass slide) and total RNA is amplified, labeled and introduced into the
hybridization solution. There may be tens of thousands of spots on an array,
each containing a huge number of identical single stranded DNA molecules of
lengths from twenty to hundreds of nucleotides. The spot diameter is of the
order of 0.1 mm or smaller. The identity of each spot is known by its position.
The whole array occupies no more than a few dozen square centimeters. For
gene expression studies, probes from one spot ideally should identify (be
complementary to) one gene or one exon in the genome. In practice this is
not always so simple and may not even be generally possible due to families
of similar genes in a genome.
In microarrays, the probes are oligonucleotides, cDNA or small fragments
of PCR products that correspond e.g. to mRNAs. The probes are synthe-
sized prior to deposition on the array surface and are then “spotted” onto the
surface. Arrays can also be produced by printing desired sequences by synthe-
sizing directly onto the array surface instead of depositing intact sequences.
Printed sequences may be longer (60-mer probes such as the Agilent design)
or shorter (25-mer probes produced by Affymetrix). Although oligonucleotide
probes are often used in “spotted” microarrays, the term “oligonucleotide ar-
ray” most often refers to “printed” array.
In microarrays dedicated to expression studies, probes are complementary
to sequences of known or predicted genes, to their transcribed regions (pref-
erentially to 3’UTRs, as a regions more divergent even in families of highly

(a)

(b)

Fig. 9.3 Types of genomic tiling arrays: covering almost the whole (a) or the whole
(b) genomic sequence
9.2 Analysis of Gene Expression at RNA Level 145

similar genes). There are also commercially available arrays dedicated to mi-
croRNA. Furthermore, DNA microarrays can densely cover a whole genome
or a genomic region of interest. They are called genomic tiling arrays (Fig.
9.3). In such arrays usually oligonucleotides are used. To cover a mammalian
genome several arrays are necessary (e.g. to interrogate the mouse genome,
Affymetrix offers the set of seven arrays containing approximately 45 million
oligonucleotide probes). Tiling arrays dedicated to studies of transcription
factors interactions with DNA (by ChIP-chip; ch. 9.3) are a single arrays. For
example Affymetrix Promoter Array is comprised of over 4.6 million probes
(25 bp long) tiled to interrogate over 25,000 known or predicted promoter
regions. Each promoter region covers approximately 6 kb upstream through
2.5 kb downstream of 5’ transcription start sites.
Oligonucleotide microarrays can be used to detect point mutations (the
missing, adding, or changing of a single base) in a known DNA sequence. Sin-
gle base mismatches do have much more influence on binding to an oligonu-
cleotide sequence compared to much longer cDNA. For example, a small
genome can be loaded on a chip as a set of thousands of 20-25 bp long
fragments. When a single base pair match exists, the fluorescence intensity
decreases significant. This technique gives possibilities to find most of the
point mutations (or SNPs) in a known DNA sequence.

9.2.1.1 Two-Color Microarrays

One of the most popular microarray applications allows the comparison of
two different samples (e.g. RNA from the same cell type in a “healthy” and
“diseased” state) hybridizing them on one array (Fig. 9.4). The total mRNA
from the cells in two different conditions is extracted and reverse transcription
PCR (RT-PCR) is used to convert the transcripts into cDNA. The cDNAs
are usually composed of 500 -2,000 base pairs long. They have to be amplified
and labeled with two different fluorophores (commonly used are cyanine 3-
or cyanine 5-labeled CTP: abbreviated to Cy3 and Cy5, respectively). Nu-
cleotides labeled with a red fluorescent dye Cy5 can be incorporated into the
experimental cDNA (e.g. from cells in a “diseased” state), while nucleotides
labeled with a green fluorescent dye Cy3 - into the control cDNA (e.g. from
cells in a “healthy” state). Both samples are mixed and after fragmentation
allowed to hybridize overnight to the array. Excess hybridization buffer is
washed off and the slides are then scanned. Labeled molecules (in this case -
products of gene expression in examined cells) hybridize to their complemen-
tary sequences in the spots due to the preferential binding - complementary
single stranded nucleic acid sequences tend to attract to each other and the
longer the complementary parts, the stronger the attraction. Such strategy
allows to estimate in which sample the corresponding sequence is more abun-
dant, however it is not suitable for measurement of the expression level in
examined samples.
146 9 High-Throughput Technologies in Molecular Biology

Fig. 9.4 A diagram of a typical two-color microarray experiment. Only a small

fragment of the array is shown: position of spots in which more experimental (red)
sample has been hybridized is marked by white squares. This means that in the
experimental sample the corresponding sequence is more abundant than in control
sample. When corresponding sequences are in equal amount in both samples -
a yellow spot is observed.

Two-color microarrays are commercially available (e.g. “Dual-Mode” plat-

form from Agilent, “DualChip” platform from Eppendorf, and “Arrayit” from
TeleChem International). Arrays may also be easily customized for each ex-
periment and printed on a smaller scale.

9.2.1.2 One-Color Microarrays

One-color (single-channel) microarrays, i.e. the arrays designed for hybridiza-
tion with one labeled sample, provide intensity data for each probe indicating
a relative level of hybridization with the labeled target. They do not truly
indicate abundance levels of gene expression but rather relative abundance
when compared to other samples or conditions when processed in the same
experiment. Among popular single-channel systems are the Affymetrix “Gene
Chip”, Illumina “Bead Chip”, Agilent single-channel arrays, the Applied Mi-
croarrays “CodeLink” arrays, and the Eppendorf “DualChip & Silverquant”.
The procedure of sample preparation for hybridization on one-color array
is essentially the same as for two-color array. For expression analysis RNA
has to be reverse transcribed, amplified and labeled. After fragmentation the
sample is hybridized overnight to the array, washed, stained and scanned.
Obtained hybridized microarray images contain the raw data (Fig. 9.5).
9.3 Studies of Gene Expression Regulation by Chromatin Immunoprecipitation147

To obtain information about gene expression each spot on the array should be
identified, its intensity measured and normalized to make data from different
arrays comparable.

Fig. 9.5 An example of the raw data obtained from Affymetrix one-color array (in
this example - from Promoter Tiling Array). From left to right larger magnifications
are shown. White spots visible on the largest magnification represent individual
probes which hybridized with labeled sample.

9.3 Studies of Gene Expression Regulation by

Chromatin Immunoprecipitation
The protein-DNA interactions play a crucial role in many cellular processes
such as DNA replication, recombination, repair, or gene transcription. The
most informative are studies of interactions that take place in vivo, in liv-
ing cells. They can be studied by chromatin immunoprecipitation (ChIP;
Fig. 9.6). ChIP is a powerful method used to determine the location of DNA
binding sites on the genome for a particular protein of interest at a given
moment. It is possible only if a specific antibody that can recognize protein
of interest is available (immunoprecipitation is the technique of precipitating
a protein antigen out of solution using an antibody that specifically binds to
that particular protein). Proteins that are strongly bound to DNA (like his-
tones) can be immunoprecipitated without cell fixation. Weakly bound pro-
teins (including transcription factors) have to be cross-linked to the DNA that
they are binding. The crosslinking is often performed by applying formalde-
hyde to the cells (or tissue). Following crosslinking, the cells are lysed and
the chromatin is physically (ultrasound) or enzymatically (nucleases) bro-
ken into small pieces (0.2-1 kb in length); the shorter chromatin fragments
the better mapping of protein binding sites could be obtained. Next, the
immunoprecipitation by a specific antibody is done. In parallel, a control
reaction is also carried out: with unspecific antibody or without antibody.
Antibody-protein-DNA complexes are extracted from solution using bacte-
rial protein A or protein G (that specifically binds antibodies) immobilized on
agarose or magnetic beads. Samples have to be washed to remove an excess of
148 9 High-Throughput Technologies in Molecular Biology

Fig. 9.6 Principles of chromatin immunoprecipitation (ChIP)

material and protein-DNA complexes are purified. Complexes are then heated
to reverse the formaldehyde cross-linking, allowing the DNA to be separated
from the proteins.
The identity and quantity of the isolated DNA fragments (both experi-
mental and negative control) can then be determined by PCR (ch. 4.1.2).
The limitation of ChIP-PCR is that one must have an idea which genomic
region is being targeted in order to design the correct PCR primers. Instead
of PCR, a DNA microarray (ch. 9.2.1) can be used (ChIP-chip) what allows
to find on a genome-wide scale where the protein binds. The limitation of
ChIP-chip is that not all target sequences could be present on the array.
Alternatively, ChIP can be combined with sequencing (ChIP-seq). In fact,
recently emerged massive parallel sequencing (ch. 9.1.2) can localize protein
binding sites (ChIP products) in a high-throughput, cost-effective fashion.
ChIP-seq allows to find out all genomic targets of proteins of interest, also
these that are far away of the promoters. By integrating a large number of
short reads from sequencer, highly precise (± 50 bp) binding site localization
is obtained. Obviously, these sequences have to be mapped to the genome.
9.4 Analysis of Protein Expression 149

9.4 Analysis of Protein Expression

It is now known that mRNA level does not always correlate with protein
content. Thus, after genomics and transcriptomics, proteomics is considered
the next step in the study of biological systems. Proteomics can be defined
as the qualitative and quantitative comparison of proteomes under different
conditions to further unravel biological processes.
An organism’s genome is quite constant (although mobile elements could
introduce some differences even between cells of one organism). The proteome
is much more complicated than the genome, mostly because it differs from
cell to cell and from state to state - distinct genes are expressed in distinct cell
types and after different stimuli. What’s more, many proteins are also sub-
jected to a wide variety of chemical modifications after translation (phospho-
rylation, ubiquitination, methylation, acetylation, glycosylation, etc.). Some
proteins undergo several modifications, often in time-dependent combina-
tions. The human genome is estimated to encode between 20,000 and 25,000
non-redundant proteins. However, the number of unique protein species likely
increase by order of magnitude due to alternative maturation of transcripts
(e.g. RNA splicing), proteolysis, and post-translational modifications. It il-
lustrates the potential complexity one has to deal with when studying protein
structure and function.
Proteins are responsible for all cellular functions. Thus, characterizing of
the proteome brings the most reliable information about the actual and cur-
rent state of a living biological system. Classically, antibodies to particu-
lar proteins or to their modified forms are used in biochemistry and cell
biology studies. They are employed in such (not described here) protein-spec-
ific techniques as Western blotting, immunohistochemistry, immunoprecipita-
tion, ELISA, and others. More recently methods of mass spectrometry, which
are not limited to specific antibody-antigen interactions, are implemented as
a standard analytical tool for characterization of proteomes.

9.4.1 MALDI Mass Spectrometry

Mass spectrometry (MS) is an analytical technique that measures the mass-
to-charge ratio (m/z) of charged particles (not only peptides or proteins).
This allows to establish a molecular weight of the analyzed molecule. In
proteomics, by using different techniques of mass spectrometry, the molecu-
lar weights of specific peptides and proteins in complex mixtures containing
just picomoles of particular molecules can be read. The practical accuracy of
measurement of molecular weights from mass spectra usually reaches 0.01%,
while in the case of electrophoretic techniques such accuracy is somewhere
between 5 and 10%. A typical mass spectrometer consists of an ion source,
150 9 High-Throughput Technologies in Molecular Biology

the analyzer, and detector, combined with computerized control system for
recording and analysis of data. Depending on a type of the material under
analysis (the analyte) various techniques of ionization and separation of ions
in the analyzers are used. Two ionization methods: matrix-assisted laser(-in-
duced-)desorption ionization (MALDI) and electrospray ionization (ESI) are
the most commonly used for protein analyses.
MALDI spectrometers are used for the analysis of proteins and peptides
with molecular weights ranging from 400 Da to as much as several hundred
thousand Da. In the MALDI spectrometers the analyte molecules (e.g., prot-
ein or peptide) are co-crystallized with matrix molecules that absorb UV light
(cinnamic acid derivatives are the most frequently used for protein studies).
As a result of irradiation of a matrix and analyte molecules mixture by a nano-
second laser pulse, protonated molecules of the analyte are desorbed from the
crystals (matrix molecules serve as a source of protons). Most of the laser en-
ergy is absorbed by the matrix what prevents unwanted fragmentation of the
analyte molecules, which are usually endowed with a single protons and form
[M+H]+ molecular ions. Similar principle of ionization is used in Surface-
Enhanced Laser Desorption Ionization (SELDI) spectrometers. In this type
of spectrometers the laser beam-induced desorption of analyte molecules oc-
curs from different types of surfaces, which specifically bind different fractions
of proteins (such surfaces are coated with substances which are equivalent to
chromatography deposits).
MALDI (and SELDI) spectrometers are usually coupled with Time of
Flight (ToF) ion analyzers, which use strong electric field to accelerate the

Fig. 9.7 Schematic representation of MALDI-ToF mass spectroscopy

9.4 Analysis of Protein Expression 151

ions. The accelerated ions are then introduced into the high vacuum flight
tube and continue to fly until they reach the detector (ions should be acceler-
ated to the same kinetic energy to ensure that all ions of identical mass move
at the same speed). Light ions reach the detector earlier than heavy ions.
Registered time of flight of analyzed molecular ions (typically ranging from
0.01 to 1 ms) is inversely proportional to the m/z value and can be converted
into actual mass of the analytes. Resulting data are represented in a form of
spectra (Fig. 9.7).
The resolution and mass accuracy are dependent on the time window
wherein ions of the same mass reach the detector. This is for a large part
determined by the start velocity distribution. To correct start velocity distri-
bution the reflectron mode could be used. In this mode, the drift direction of
the ions in an electric counter field is reversed (Fig. 9.7). Ions of the same mass
but higher start energy drift deeper into the reflectron before being reflected
and fly a longer distance before they reach the detector. In this way, they
catch up with the slower moving ions at a certain point after the reflectron.
The detector is located at this focusing point. The reflectron greatly en-
hances resolution by correcting start velocity distribution and by prolonging
the flight time. Although use of the reflectron mode results in higher resolu-
tion, reflection reduces sensitivity and high molecular weight (poly)peptides
can only be detected in linear mode.
The described method in its basic version allows the identification of char-
acteristic features of the protein profiles of analyzed mixtures. However,
MALDI-ToF spectrometry also allows the identification of proteins based
on the amino acid sequence in polypeptide chains. Most typically, the an-
alyzed protein is processed enzymaticaly (e.g., by trypsin digestion), then
selected peptides are further fragmented in the spectrometer, and sizes (m/z
values) of fragmentary ions are determined in the so called tandem mass
spectrometry (MS/MS). On the basis of size of fragmentary ions the ap-
propriate computational analysis allows the establishing of the sequence of
amino acids in the peptide chain and the identification of corresponding pro-
tein. Additionally, MALDI-ToF MS can be applied directly to samples of
biological tissues. While traditional methods of protein analysis require the
homogenization of whole tissues, direct MS analysis of tissue requires much
less sample manipulation and maintains the spatial integrity of the specimen.
Thus, a single section of a tissue sample could be used not only for identifica-
tion of proteins but also for determination of spatial distributions of detected
molecules. Spatially-correlated MS analysis of a tissue sample that can reveal
abundance of different biomolecular ions in different sample spots is called
imaging mass spectrometry (IMS).
Proteomics studies generate high-dimensional spectral data sets where tra-
ditional statistical and computational methods are not sufficient for analyses,
hence novel “non-standard” mathematical approaches are frequently required.
A typical proteomic analysis of MS data consists of three steps: (i) pre-
processing of mass spectra data, (ii) identification of spectral components,
152 9 High-Throughput Technologies in Molecular Biology

(iii) statistical analyses and classification based on identified components.

The first step includes: “smoothing” of the spectrum, removing the baseline,
normalization and averaging of technical replicates. The simplest method
of spectra “smoothing” is averaging among several neighboring points. The
baseline correction flattens specific noise named baseline. Interpolation is
performed to standardize points on the m/z axis among all spectra. Normal-
ization is used for scaling the spectra - the most frequently used algorithms
base on total ion current (TIC) value or constant noise. All these procedures
reduce dimensionality of data and improve quality of further analyses. There
are several algorithms accepted for these procedures and most of proteomics
studies used comparable methods for spectra pre-processing. However, there
is no single protocol generally accepted as the standard. In fact, some of
the key parameters are tuned and adjusted for particular analyses, and type
and order of pre-processing steps may significantly differ among different
analyses. There are two general types of approaches allowing comparison of
multiple spectra. One of them bases on comparing the signal in successive
measurement points (called point-to-point analysis). The second one uses
identification and matching of spectral components. This approach usually
starts from peak detection. The aim of peak detection procedures is to find
peaks describing the composition of spectra, which is followed by alignment of
peaks identified in different spectra. Most of algorithms used for peak detec-
tion identify local maxima and minima; local maxima are classified as peaks
only when they have signal to noise ratio above a given threshold. Alterna-
tive procedure for identification of spectral components bases on modeling
of spectra as a sum of Gaussian components, then the model is adjusted to
experimental data. Such procedure avoids artifacts linked with the methods
of point-to-point and peak alignment, and facilitates quantitative and statis-
tical analysis. The objective of the last step of spectral data analysis is to find
patterns in the data sets and to classify samples. Many of the statistical and
analytical algorithms and tools developed for functional genomics are being
used in proteomics. Different combinations of unsupervised and supervised
clustering, machine learning, pattern recognition, statistical analysis, model-
ing techniques, and database mining are used in the field. Some researchers
use custom-build protocols and algorithms while others base on commercially
available software for spectra analysis. However, all these computational tools
have to be tuned and optimized for specific applications and datasets, which
apparently add diversity to these protocols.

Acknowledgments. This work was inspired by Prof. Andrzej Polański and

supported by the Polish National Science Centre (UMO-2011/01/B/ST6/
06868) and by the European Community from the European Social Fund within
the INTERKADRA project (UDA - POKL-04.01.01-00-014/10-00).
References 153

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