INTRODUCTION TO

MOLECULAR BIOLOGY
Amb. Samson Balo, BSc., Msc. Candidate
Department of Biology Sciences
College of Science and Technology
Africa Scholar Resource Institute University
Introduction to Molecular Biology
• The biochemistry of genes and their products

• Molecular biology; the branch of biology that

study gene structure and func on at the molecular
level

• The Molecular biology field overlaps with other

areas, par cularly gene cs and biochemistry

• The Molecular biology allows the laboratory to be

predic ve in nature; events that occur in the
future.
ORIGIN OF THE MOLECULAR BIOLOGY

• Term was coined by Rockefeller founda on in

1938
• Later the term was used by William Asbury to
study chemical and physical structure of
biological molecules
• Three areas contributed to the development:
Ø Instrumenta on and techniques
Ø Radioac ve and fluorescent labelling
Ø Nucleic acids and enzymology
 Three Domain of Life

Eukaryotic
Prokaryotic
Archaea
 Two Cell Types
Prokaryo c Cells
Eukaryo c Cells
• Three Domains
• Defined by cell type
1.Eukarya
• Plantae
• Fungi Eukaryotic
• Animalia
• Pro sta
2.Bacteria
3.Archaea Prokaryotic
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5
 2 The Genetic Material
Ø The genetic material of the cell is the Deoxyribonucleic acid (DNA).
Ø Key features of the DNA include:
§ Able to store information used to control both the development and the metabolic
activities of cells;

§ Stable so it can be replicated accurately during cell division and be transmitted for
generations; and,

§ Able to undergo mutations thus providing the genetic variability required for evolution.

Ø Deoxyribonucleic acid (DNA) is a nucleic acid molecule that carries the genetic
instructions used in the growth, development, functioning and reproduction of all
living organisms. 12/11/22
3 Evidences for DNA as Genetic Material
Ø The idea that genetic material is nucleic acid had its roots in the discovery of
transformation in 1928 by Frederick Grifth.
Ø Grifth laid the foundation with his experiments on transformation in the bacterium
pneumococcus, now known as Streptococcus pneumonia.
Ø He was trying to make a vaccine against pneumonia and instead discovered
transformation.
Ø Grifth observed that live S bacteria could kill mice injected with them.
Ø When he heat killed the S variants and mixed them with live R variants, and then
injected the mixture in the mice, they died.
Ø Grifth was able to isolate the bacteria from the dead mice, and found them to be
of the S variety.
Ø Thus the bacteria had been Transformed from the rough to the smooth version.

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4 Evidences for DNA as Genetic Material

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5 Avery, MacLeod, McCarty Experiment
Ø Although Grifth discovered transformation, his experiment did not show the
chemical nature of the transforming substance.
Ø Oswald Avery, Colin MacLeod, and Maclyn McCarty supplied the missing piece
(chemical nature of the transforming substance) in 1944.
Ø Using a similar transformation test, they were able to show that the transforming
substance was DNA.
Ø When they added DNA isolated from S cells to growing cultures of R cells, they
observed transformation: A few cells of type S cells were produced.
Ø The transforming activity was not altered by treatments that destroyed either protein
or RNA.
Ø However, treatments that destroyed DNA eliminated the transforming activity.
Ø The experiments thus implied that the substance responsible for genetic
transformation was the DNA – hence DNA is the genetic material.

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6 Avery, MacLeod, McCarty Experiment

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7 Hershey and Chase experiment
Ø Performed in 1952, using bacteriophage, a type of virus that infect bacteria.
Ø The phage are made up of equal parts of protein and DNA.
Ø It was known that the phage infect by anchoring the outer shell to the cell surface
and then deposit the inner components to the cell, infecting it.
Ø Using radioactive isotopes, phage were labeled with either 35# (sulfur) to label
proteins or 32% (phosphorus) to label DNA.
Ø phage were incubated with bacteria to allow infection, and then shaken off the
bacteria.
Ø centrifugation then separated the bacteria into the pellet, with phage in the
supernatant found that 35# stayed with the phage, while 32% was with the bacteria.
Ø Alfred D. Hershey and Martha Chase concluded that phage injected DNA into
bacteria to infect them.

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8 Hershey and Chase experiment

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9 Structure of DNA
Ø Is the “blueprint” of an organism.
Ø Composed of small molecules called nucleotides.
Ø Nucleotide is a collection of three “building blocks” (pentose sugar, nitrogenous
bases & phosphate group).
Ø Nucleoside: ve carbon + nitrogenous base
Ø Has four bases: adenine (A), cytosine (C), guanine (G) and thymine (T)
Ø The adenine pair with thymine and the guanine pair with cytosine.
Ø nucleotides are linked by a 3’, 5’ phosphodiester linkage resulting chain that has a
5’ end and a 3’ end.
Ø The phosphates and sugars are collectively called the “backbone” of the strand.
Ø The sugar and base are linked by a glycosidic bond.

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10 Structure of DNA

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11 Structure of DNA

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12 Structure of DNA
Ø There are two kinds of nitrogenous bases: the nine-membered double-ringed
purines and the six-membered single-ringed pyrimidines.
Ø There are two types of purines: Adenine(A) and Guanine(G).
Ø There are also three types of pyrimidines: Thymine(T), Cytosine(C), and Uracil (U)
(found in RNA only).
Ø DNA strands are antiparallel which means, in a double helix, the direction of the
nucleotides in one strand is opposite to their direction in the other strand.
Ø Each type of nucleotide base on one-strand bonds with just one type of
nucleotide base on the other strand – an arrangement called complementary
base pairing.
Ø Bases are bonded to each others by hydrogen bonds where:
o A and T (2 bonds)
o C and G (3 bonds)

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13 Complementary Base Pairing

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14 Watson-Crick Model
Ø The accepted model for the structure of the DNA double helix was published by
James Watson and Francis Crick in 1953 .
Ø DNA was shown as:
§ double helix with antiparallel strands
§ each strand a nucleotide chain held together by phosphodiester linkages
§ strands held together by hydrogen bonds between the bases (basepairs)
§ A paired with T, with 2 hydrogen bonds
§ C paired with G, with 3 hydrogen bonds
Ø The double-helix model strongly suggested a way to store information in the
sequence of bases, which indeed appears to be true.
Ø The determination of the DNA structure by Watson and Crick is considered the
major landmark of modern biology.

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15 Nucleic Acid – RNA
Ø Its structure is similar to the structure of DNA
Ø It contains sugar + phosphate + base
Ø However, the sugar is a ribose (instead of deoxyribose)
Ø Contains four bases:
§ Adenine (A)
§ Guanine (G)
§ Cytosine (C)
§ Uracil (instead of Thymine T)
Ø RNA is single-stranded unlike DNA
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16 Types of RNA
Ø The three main types are:
§ Ribosomal RNA (rRNA): functions in the reading of the order of amino acid and
linking amino acids together during protein synthesis.
§ Transfer RNA (tRNA): functions in carrying amino acids to the ribosome to be
assembled into proteins.
§ Messenger RNA (mRNA): carries the information specifying the amino acid
sequence of a protein to the ribosome.
Ø Other small types include:
§ Heterogeneous nuclear RNA (hnRNA or pre-mRNA): is the precursor of mRNA,
formed during its post-transcriptional processing.
§ Small nuclear RNA (snRNA): only found in the nucleus of eukaryotes and
participate in splicing (removal of introns) mRNA.
§ Micro-RNA: short, non-coding RNA that control the expression or repression of
other genes during development. 12/11/22
17 The Central Dogma
Ø The central dogma of molecular biology is that DNA is transcribed to RNA
which is translated to protein.
Ø DNA (genetic information in genes) ® RNA (copies of genes) ® proteins
(functional molecules).
Ø This ow of information is unidirectional and irreversible (except for some
viruses).
Ø The information carried within the DNA dictates the end product (protein)
that will be synthesized.
Ø This information is the genetic code.
Ø Conversion of DNA encoded information to RNA is called transcription.
Ø The information from a mRNA is then translated to an amino acid sequence
in the corresponding protein.
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18 DNA Replication
Ø It is the process of copying a DNA molecule.
Ø Each old DNA strand of the parent molecule serves as a template for a new strand in a
daughter molecule.
Ø DNA replication is semi-conservative because one of the two old strands is conserved,
or present, in each daughter molecule.
Ø Replication requires the following steps:
§ Unwinding: the old strands that make up the parent DNA molecule are unwound and
“unzipped” by special enzyme called helicase. Special proteins called single-strand binding
proteins keep it open.
§ Complimentary base pairing: new complimentary nucleotides, always present in the nucleus,
are positioned by an enzyme complex called DNA polymerase. This is done through a process
called complimentary base pairing.
§ Joining: the complimentary nucleotides join to form new strands. The joining process is carried
out by DNA polymerase. DNA polymerase also proofread the newly synthesized strands and
correct all errors.
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19 Replication: Prokaryotes vs Eukaryotes
Ø Similarities:
§ Both are bi-directional processes
§ DNA polymerases work 5’ to 3’
§ Primers are required
§ Initiator proteins/enzymes bind at the origin of replication
Ø Differences:
§ Replication in prokaryotes starts at a single origin of replication while
eukaryotes have multiple origins of replication.
§ The rate of replication in prokaryotes is faster (10×) than in eukaryotes
§ The number of DNA polymerases in prokaryotes is less than that in
eukaryotes.
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20 Pattern for DNA Replication

Ø Three alternative patterns of DNA replication were proposed:

§ semiconservative replication pattern suggested that each strand
could serve as a template for making a complementary strand with
one strand old and one new.
§ Dispersive replication results in daughter duplexes that consist of
strands containing only segments of parental DNA and newly
synthesized DNA
§ In conservative replication, one daughter duplex consists of two newly
synthesized strands, and the parent duplex is conserved
Ø Experiments with E. coli supported the semiconservative
replication pattern.

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21 Pattern of Replication

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22
Transcription

Ø Process by which the genetic information is conveyed from a double stranded

DNA molecule to a single stranded RNA molecule.
Ø Only one strand of DNA serves as a template - the anti-sense strand.
Ø The complementary strand, called sense strand, has a sequence identical to the
RNA sequence (except for a U in place of a T).
Ø The process is divided into three stages in both prokaryotes and eukaryotes:
§ Initiation: the promoter sequence attracts RNA polymerase to begin transcription at a
site specied by the promoter; RNA polymerase unwind the double strand.
§ Elongation: RNA polymerase creates a bubble that moves down DNA at constant rate
leaving growing RNA strands protruding from the bubble. The synthesis is done in the
5’-3’ direction while the template strand is in the 3’-5’ position
§ Termination: here sequence of nucleotides terminate transcription and this sequence
is known as the termination sequence.
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23
Transcription cont.
Ø Salient features of transcription include:
§ RNA polymerase:
– catalyzes the addition of one ribonucleotide at a time,
– extending the RNA strand being synthesized in the 5’ to 3’ direction.
§ Promoter:
– DNA sequences near the beginning of a gene.
– These signal the RNA polymerase to begin transcription.

§ Terminators:
– sequences within the RNA products,
– which signal the RNA polymerase to stop transcription.

Ø Transcription is similar to DNA synthesis except:

− The precursors are NTPs (not dNTPs).
− No primer is needed to initiate synthesis.
− Uracil is inserted instead of thymine
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24 Transcription: Eukaryotes Vs Prokaryotes
Ø Eukaryotic transcription differs from that of prokaryotes by having:
§ Three RNA polymerase enzymes (RNAP I, RNAP II, and RNA III)
§ Initiation complex form at the promoter
§ RNAs are modied after transcription.
Ø Prokaryotes have a single RNA polymerase (which has subunits - a, b and b’)
that can bind to the DNA template directly during the transcription process.
Ø The eukaryotic RNA-pol requires co-factors to bind to the DNA template
together in the transcription process.
Ø In eukaryotes, newly synthesized mRNA are processed before transported to
the cytoplasm for translation to occur.
Ø The processing steps include:
– Addition of a 5’ 7-methyl guanosine cap (capping).
– Addition of a poly-A tail at the 3’ end (polyadenylation)
– RNA splicing to remove intervening sequences (remove introns).

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25 Transcription

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26 Translation
Ø It is conversion of a messenger RNA sequence into amino acid sequence
in order to transmit information.
Ø mRNA carries the genetic code to cytoplasm, where it is translated by
ribosomes.
Ø Ribosomal RNA forms an integral part of the ribosome, without which
translation would not be possible
Ø Ribosomes have two subunits – small and large – that come together to
make proteins.
Ø Transfer RNA collects the proper amino acids and bring them to the
ribosomes where they are used to synthesize the polypeptide chain.

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27 Translation cont.
Ø Translation involves three steps:
§ Initiation: all proteins begin with a methionine start codon (AUG) that
signals initiation of protein synthesis. Initiation factors help the mRNA to
attach to the ribosomes forming a complex.
§ Elongation: Once the ribosome complex has been formed and the
tRNA carrying the methionine is in the P-site, the ribosome can now
begin sliding along the mRNA as tRNA add new amino acids.
§ Termination: synthesis continues until a stop codon is reached, which
bind release factor proteins. If the ribosome reads either any of the
stop codon (UAA, UGA or UAG), a protein called release factor will
bind to A-site thus stopping the process.

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28 Prokaryotic translation Vs Eukaryotic translation
Ø Although the process is similar, the component of eukaryotic
and prokaryotic translation can not be mixed.
Ø Ribosomes:
´ The ribosomes of prokaryotes (30S, 50S) are smaller in sizes than those of
eukaryotes (40S, 60S)
´ S stand for Svedberg unit – a unit that measure the rate at which a given
particle or molecule sediments.
Ø Soluble translation factors:
´ There are differences in the Initiation factors (IF), elongation factors (EF)
and termination factors (TF) of prokaryotes and eukaryotes.
´ For instance, the termination factors in prokaryotes are RF1, RF2, and RF3
while those for eukaryotes are TF1, TF2, TF3.
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29 Translation

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30 The Genetic Code

Ø It is a set of rules that translate mRNA into the amino acids sequence of protein.
Ø Important elements of the genetic code include:
1. The code is a triplet code: Each mRNA codon (word) that species a particular amino acid
in a polypeptide chain consists of three nucleotides (letters). For example, AAG = lysine
2. The code is non-overlapping: The mRNA encoding one protein is read in successive groups
of three nucleotides.
3. The code is degenerate: More than one mRNA codon (word) occurs for some amino acids
(ie. AAG and AAA are read as both read as lysine)
4. There is more than one tRNA type (therefore more than one anticodon) for some amino
acids.
5. The code has start signals (AUG and rarely GUG) and stop signals (UAA, UAG, and UGA).
6. The code is commaless.
7. The code is almost universal.

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31 The Genetic Code

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32 Gene Expression Regulation
´ Gene expression is the process by which the genetic code - the nucleotide
sequence - of a gene is used to direct protein synthesis and produce the
structures of the cell.
´ The process involves two main stages – transcription and translation.
q Gene Regulation in Prokaryotes:
§ Gene regulation is the controls that act on gene expression.
§ Gene regulation is important because gene expression is expensive and
inappropriate gene expression can be harmful to cells/organisms
§ Also, it takes a lot of energy to make RNA and protein.
§ In prokaryotes, genes are regulated using an operon.
§ Operon – group of genes that are transcribed together and code for
functionally similar proteins.
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33 Gene Regulation in Prokaryotes
´ An operon consist of:
´ Promoter – section of DNA where RNA polymerase binds
´ Operator – Controls activation of transcription
´ On/off switch between promoter and genes for proteins – structural
genes
´ Repressor protein – binds to operator to block RNA polymerase
and shut down transcription.
´ Turns off the operon
´ Corepressor – keeps the repressor protein on the operator
´ Inducer – pulls repressor off the operator
´ Turns on the operon – lactose on the lac operon
´ Regulatory gene – produces the repressor protein
´ Structural genes – code for proteins
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34 Positive and Negative Gene Regulation

´ Negative control:
´ Repressible: usually on but can be inhibited; eg. trp operon, allosteric inhibition,
tryptophan present prevents its own production (anabolic).
´ Inducible: usually off, but can be turned on; an inducer (a specic small molecule,
allolactose in the lac operon) inactivates the repressor and allows transcription
(catabolic).
´ Positive control:
´ E. coli prefer to use glucose for energy, they will only use lactose when glucose is in short
supply
´ glucose cAMP binds to regulatory protein “CAP” (Catabolite activator protein) &
stimulates gene transcription, thus a positive gene regulation.
´ The cAMP & CAP combination allow RNA polymerase to bind to the promoter sequence
more efciently.
´ Remember cAMP is regulating the gene expression in the bacteria.

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35 Gene Regulation in Prokaryotes

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36 Gene Regulation in Eukaryotes
Ø Eukaryotic gene regulation is more complex because
eukaryotes possess a nucleus.
Ø Two “ categories” of eukaryotic gene regulation exist:
§ Short-term - genes are quickly turned on or off in response to
the environment and demands of the cell.
§ Long-term - genes for development and differentiation.
Ø Eukaryotic gene expression is regulated at six (6) levels:
1. Transcription
2. RNA processing
3. mRNA transport
4. mRNA translation
5. mRNA degradation
6. Protein degradation

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37 Gene Regulation in Eukaryotes
Ø Transcriptional regulation: This is accomplished by promoters and
enhancers.
§ Promoters: they can be positively or negatively regulated thus regulating
transcription.
§ Enhancers: their interactions with regulatory proteins determine if transcription is
activated or repressed.
Ø RNA processing: regulates mRNA production from precursor RNAs.
§ Two independent regulatory mechanisms occur:
o Polyadenylation: where the poly A tail is added
o Splicing: where exons are splice.
Ø mRNA transport: Eukaryote mRNA transport from the nucleus to the
cytoplasm can be regulated.
Ø mRNA translation: some proteins can inhibit translation thus regulating gene
expression.
Ø mRNA degradation: all RNAs in the cytoplasm are subject to degradation.
Ø Protein degradation: Proteins can be short-lived (e.g., steroid receptors)
or long-lived (e.g., lens proteins in your eyes).
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38

´ The rst step to knowledge is to know we are ignorant.

- Lord David Cecil

´ You’re going to make mistakes in life. It’s what you do after the mistakes that counts.
- Brandi Chastain

THE END!!

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