Genes that do not code proteins also are transcribed:

1. rRNA, ribosomal RNA

 Catalyze protein synthesis by facilitating the binding of tRNA

(and their amino acids) to mRNA.

2. tRNA, transfer RNA

 Transport amino acids to mRNA for translation.

3. snRNA, small nuclear RNA

 Combine with proteins to form complexes used in RNA

processing (splicosomes used for intron removal).
 Synthesis of ribosomal RNA and ribosomes:
1. Cells contain thousands of ribosomes.

2. Consist of two subunits (large and small) in prokaryotes and eukaryotes, in combination
with ribosomal proteins.

3. E. coli 70S model:

 50S subunit = 23S (2,904 nt) + 5S (120 nt) + 34 proteins

 30S subunit = 16S (1,542 nt) + 20 proteins

4. Mammalian 80S model:

 60S subunit = 28S (4,700 nt) +5.8S (156 nt) + 5S (120 nt) + 50 proteins

 40S subunit = 18S (1,900 nt) + 35 proteins

5. DNA regions that code for rRNA are called ribosomal DNA (rDNA).

6. Eukaryotes have many copies of rRNA genes tandemly repeated.

Synthesis of ribosomal RNA and ribosomes (continued):
7. Transcription occurs by the same mechanism as protein-coding
genes, but generally using RNA polymerase I.

8. rRNA synthesis requires its own array transcription factors (TFs)

9. Coding sequences for RNA subunits within rDNA genes contain

internal (ITS), external (ETS), and nontranscribed spacers (NTS).

10. ITS units separate the RNA subunits through the pre-rRNA stage,
whereupon ITS & ETS are cleaved out and rRNAs are assembled.
11. Subunits of mature ribosomes are bonded together by H-bonds.

12. Finally, transported to the cytoplasm to initiate protein synthesis.

Mammalian example of 80S rRNA
 Synthesis of tRNA:
1. tRNA genes also occur in repeated copies throughout the genome, and
may contain introns.

2. Each tRNA (75-90 nt in length) has a different sequence that binds a

different amino acid.

3. Many tRNAs undergo extensive post-transcription modification,

especially those in the mitochondria and chloroplast.

4. tRNAs form clover-leaf structures, with complementary base-pairing

between regions to form four stems and loops.

5. Loop #2 contains the anti-codon, which recognizes

mRNA codons during translation.

6. Same general mechanism using RNA polymerase III, promoters, unique

TFs, plus post-transcriptional modification from pre-tRNA.
How do nucleotides in the MRNA molecule specify the aa sequence in
proteins?

• With four different nucleotides (A, C, G, U), a three-letter code

generates 64 possible codons, yet there are only 20 different aas.
THE GENETIC CODE
• The genetic code is the collection of base sequences (codons) that
correspond to each aa and to stop signals for translation.

• Since there are 20 aas there must be more than 20 codons to

include signals for starting and stopping the synthesis of particular
protein molecules.

 In this case, several codons designate the same aa – that is, the
code is redundant or degenerate.
31-Jan-25 10
CHARACTERS OF THE GENETIC CODE

SPECIFIC OR UNAMBIGUOUS
DEGENERATE
UNIVERSALITY
NON-OVERLAPPING.
COMMA-LESS
ALL ORGANISMS SHARE THE SAME GENETIC LANGUAGE.
FOR EXAMPLE,
-LYSINE IS CODED FOR BY AAA OR AAG IN THE MRNA OF ALL ORGANISMS,
-ARGENINE BY CGU, CGC, CGA, CGG, AGA, AND AGG

31-Jan-25 11
 Second Base
U C A G
UUU UCU UAU UGU U
phe tyr cys
UUC UCC UAC UGC C
U ser
UUA UCA UAA stop UGA stop A
leu
UUG UCG UAG stop UGG trp G
CUU CCU CAU his CGU U
CUC CCC CAC CGC C

Third Base
First Base

C leu pro arg

CUA CCA CAA gln CGA A
CUG CCG CAG CGG G
AUU ACU AAU AGU ser U
asn
AUC ile ACC AAC AGC C
A thr
AUA ACA AAA AGA A
lys arg
AUG met (start) ACG AAG AGG G
GUU GCU GAU GGU U
asp
GUC GCC GAC GGC C
G val ala gly
GUA GCA GAA GGA A
glu
GUG GCG GAG GGG G
 Start codon
• In both eukaryotes and prokaryotes, AUG is usually
the start codon for protein synthesis, although in
some cases GUG is used.
– AUG codes for Methionine
– GUG normally codes for valine
• Thus, in examining the sequence of a particular
mRNA for the location of the amino acid-coding
sequence, we look near the 5’ end for the
occurrence of the AUG start codon and begin
reading the aa sequence from there.
31-Jan-25 14
The translation of an mRNA begins with the AUG codon
In bacteria: formylmethionine
In eukaryotes: methionine
and stops at: UAA, UAG, or UGA
Stop-codons move into
the ribosomal A site
(no tRNA for stop
Codons)

Release factors bind

to any ribosome with a
stop codon positioned
in the A-site
Release of the polypeptide chain,
the mRNA
and disassembly of the subunits
 Translation from gene to protein
• Components required for translation:
– mRNA
– Ribosomes
– tRNA
– Aminoacyl tRNA synthetases
– Initiation, elongation and termination factors
• The information for the proteins found in a cell is encoded in the
structural genes of the cell’s genome.
• Expression of a protein-coding gene occurs by transcription of the
gene to produce an mRNA, followed translation of the mRNA, i.e., is
the conversion of the mRNA base sequence information into an
amino acid sequence of a polypeptide.
• There are two aspects to understanding how translation occurs
i) information /coding/ aspect
– The mechanism by which a base sequence in a DNA molecule is
translated into an aa sequence of a polypeptide chain.
ii) chemical aspect
– The chemical problem refers to the actual process of synthesis of
the protein:
Processes involved in protein synthesis
• Initiating synthesis
• Linking together the amino acids in the correct order
• Terminating the chain
• Releasing the finished chain from the synthetic apparatus;
• Folding the chain; and
• Post-synthetic modification of the newly synthesized chain
31-Jan-25 17
 tRNA charging

1)Amino acid + ATP aminoacyl-AMP +

Pyrophosphate (PPi)

2)Aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP

The sum of reactions 1 and 2 is the following:

3) Amino acid + ATP + tRNA aminoacyl-tRNA

+AMP+PPi

31-Jan-25 18
31-Jan-25 20
Charging takes place in two steps: both catalyzed by the enzyme
aminoacyl-tRNA synthetase

31-Jan-25 21
The correct aa sequence is achieved as a result of:
 The specific binding of each aa to its own specific tRNA, and
 The aa specific binding between the codon of the mRNA and the
complementary anticodon in the tRNA.

How do mRNA and tRNA recognize each other?

 The specificity of codon recognition lies in the tRNA molecule and
not in the aa it carries.

THE AMINOACYL SYNTHETASE

 There is at least one and usually only one aminoacyl synthetase for
each aa.
 For a few aas specified by more than one codon, more than one
synthetase exists.
 The enzymes are clearly very specific and must be able to recognize
both a particular tRNA molecule and a particular aa.
31-Jan-25 22
• Protein synthesis occurs on ribosome
In prokaryotes
• Ribosome consists of:
• three RNA molecules
• about 52 different protein molecules – consist of
enzymatic system needed to form a peptide bond
between aas.
• A site for binding the mRNA, and
• Sites for bringing in and aligning the aas in
preparation for assembly into the finished
polypeptide chain

31-Jan-25 23
Ribosome Structure
Prokaryotic Ribosome 23S rRNA
50 S 5 S rRNA
34 proteins

30 S 16 S rRNA
70S = 2.8 million daltons 21 proteins

Eukaryotic Ribosome 28S rRNA

5.8S rRNA
60S 5S rRNA
49 proteins

40S 18S rRNA

80S = 4.5 million daltons 33 proteins
TRANSFER RNA
• tRNA carries aa to the ribosome

• tRNA molecules contain a site for amino acid attachment called

anti-codon.

• Anticodon easily recognizes the appropriate base sequence (the

codon) in the mRNA.

• There are specific tRNA molecules that correspond to each

amino acid.
• All tRNAs have the same three bases (CCA) at their
3′-ends, and the terminal adenosine is the target for charging

31-Jan-25 25
Initiation-requirements:

1. mRNA
2. Ribosome
3. Initiator tRNA (fMet tRNA in
prokaryotes)
4. 3 Initiation factors (IF1, IF2, IF3)
5. Mg2+
6. GTP (guanosine triphosphate)
Initiation-steps (e.g., prokaryotes):

1. 30S ribosome subunit + IFs/GTP bind to AUG start codon and Shine-Dalgarno
sequence composed of 8-12 purine-rich nucleotides upstream (e.g., AGGAGG).

2. Shine-Dalgarno sequence is complementary to 3’ 16S rRNA.

3. Initiator tRNA (fMet tRNA) binds AUG (with 30S subunit). All new prokaryote
proteins begin with fMet (later removed).

fMet = formylmethionine (Met modified by transformylase; AUG at all other

codon positions simply codes for Met)

mRNA 5’-AUG-3’ start codon

tRNA 3’-UAC-5’ anti-codon

4. IF3 is removed and recycled.

5. IF1 & IF2 are released and GTP is hydrolysed, catalyzing the binding of 50S rRNA
subunit.

6. Results in a 70S initiation complex (mRNA, 70S, fMet-tRNA)

Initiation codons in bacterial mRNAs are preceded by a specific sequence called a
Shine-Delgarno sequence that aligns the mRNA on the ribosome for translation by
base-pairing with a complementary sequence near the 3´ terminus of 16S rRNA.
This base-pairing interaction enables bacterial ribosomes to initiate translation,
which indicates that the secondary structure around the initiation site can
modulate the efficiency of translation of a gene. Base-pairing could therefore
occur as follows:
16S rRNA: 3’-AUUCCUCCAC-5’
INITIATION SITE: 5’-AGGAGGU-3’

31-Jan-25 30
initiation
• The large subunit of the ribosome contains
three binding sites
– Amino acyl (A site)
– Peptidyl (P site)
– Exit (E site)
• At initiation,
– The tRNAfMet occupies the P site
– A second, charged tRNA complementary to the
next codon binds the A site.

31-Jan-25 31
 P A

80 S
6 0S
e IF 1 + e IF 3
P A

e IF 1
4 0 S
e IF 3
m e th io n in e -a c y l-tR N A

G T P - e IF 2

3 'U A I 5 '

G T P - e IF 2
e IF 1
U A I
5 '
e IF 3
A T P A U G U U U G C A
5 ' 3 '
e IF 4 m R N A
A D P + P i

G T P - e IF 2
e IF 1
U A I
A U G U U U G C A
5 ' 3 ' m R N A
e IF 4 e IF 3

e IF 5
N
A
P
e IF 2 + G D P + P i
e IF 3 + e IF 1 + e IF 4
U A I
A U G U U U G C A
5 ' 3 ' m R N A
S te p s o f in it ia t io n
31-Jan-25 33
Elongation of a polypeptide:

1. Binding of the aminoacyl tRNA

(charged tRNA) to the ribosome.

2. Formation of the peptide bond.

3. Translocation of the ribosome to the

next codon.
Translation: elongation
Elongation can be thought to involve three processes:
1) aligning each aminoacylated tRNA
2) forming the peptide bond to add the new amino acid to the
polypeptide chain, and
3)moving the ribosome along the mRNA by three more bases (one
codon)
• Ribosome translocates by three bases after peptide bond formed
• New charged tRNA aligns in the A site
• Peptide bond between amino acids in A and P sites is formed;
• When the A site is filled, a peptidyl transferase activity catalyzes
the formation of the peptide bond between the amino acid in the
A site and the adjacent amino acid in the P site.
• Ribosome translocates by three more bases
• The uncharged tRNA in the A site is moved to the E site.
31-Jan-25 35
Formation of the peptide bond.

• Two aminoacyl-tRNAs positioned in the ribosome, one in the P site (5’) and
another in the A site (3’).

• Bond is cleaved between amino acid and tRNA in the P site.

• Peptidyl transferase (catalytic RNA molecule - ribozyme) forms a peptide bond

between the free amino acid in the P site and aminoacyl-tRNA in the A site.

• tRNA in the A site now has the growing polypeptide attached to it (peptidyl-
tRNA).
Translocation of the ribosome to the next codon.

• Final step of the elongation cycle.

• Ribosome advances one codon on the mRNA using EF-G (prokaryotes) or EF-2
(eukaryotes) and GTP.

• Binding of a charged tRNA in A site (3’) is blocked.

• Uncharged tRNA in P site (5’) is released.

• Peptidyl tRNA moves from A site to the P site.

• Vacant A site now contains a new codon.

• Charged tRNA anti-codon binds the A site, and the process is repeated until a
stop codon is encountered.

• Numbers and types of EFs differ between prokaryotes and eukaryotes.

• 8-10 ribosomes (polyribosome) simultaneously translate mRNA.

Termination of translation:

Signaled by a stop codon (UAA, UAG, UGA). These are called non-sense codons, or
chain-terminating codons

Stop codons have no corresponding tRNA.

Release factors (RFs) bind to stop codon and assist the ribosome in terminating
translation.

1. RF1 recognizes UAA and UAG

2. RF2 recognizes UAA and UGA
3. RF3 stimulates termination

4 termination events are triggered by release factors:

1. Peptidyl transferase (same enzyme that forms peptide bond) releases

polypeptide from the P site.

2. tRNA is released.

3. Ribosomal subunits and RF separate from mRNA.

4. fMet or Met usually is cleaved from the polypeptide.

 Post-translational Modifications
After translation, the released protein is
modified in various ways:
i. Removal of the methionine (first amino acid)
ii. Folding and assembly of proteins
iii. Protein sorting and translocation
iv. Role of Post-Translational Modifications in Sorting
and Translocation

31-Jan-25 42
31-Jan-25 43
 i. Removal of the methionine
 In eukaryotic cells, the process of protein synthesis involves translation of
messenger RNA (mRNA) into a chain of amino acids, forming a polypeptide.

 This chain typically begins with a methionine (Met) residue because the start
codon (AUG) codes for methionine.

 However, in many proteins, this initial methionine is removed as part of post-

translational modifications to produce a functional protein.

31-Jan-25 44
 ii. Folding and assembly of
proteins
 After proteins are synthesized
during translation, they must
undergo folding and assembly to
become fully functional.

 This process involves the

polypeptide chain folding into a
specific three-dimensional
structure and sometimes
assembling with other
polypeptide
31-Jan-25
chains. 45
 iii. Protein sorting /targeting/and translocation
Protein Sorting: Refers to the mechanism by which proteins are directed to their
specific cellular destinations, such as the nucleus, mitochondria, endoplasmic
reticulum (ER), Golgi apparatus, lysosomes, or plasma membrane.

Protein Translocation: Involves the actual movement of proteins across cellular

membranes to reach their target locations.

Both processes rely on specific signals within the protein sequence and recognition
by cellular machinery.

31-Jan-25 46
 Types of sorting and Translocations
Co-translational translocation:

 Protein translocation occurs while the protein is being synthesized.

 Example: Proteins with ER signal sequences are directed to the rough ER, where
they pass through the translocon channel into the ER lumen or membrane

Post-Translational Translocation:

Fully synthesized proteins in the cytosol are transported to their destinations.

31-Jan-25 47
 Sorting and Translocation in the Secretory Pathway
1. Endoplasmic Reticulum (ER):Proteins with an ER signal sequence are
translocated into the ER. Within the ER, proteins undergo initial PTMs such as
glycosylation and disulfide bond formation.

2. Golgi Apparatus: Proteins are transported to the Golgi in vesicles. Additional

PTMs, such as glycan trimming or phosphorylation, occur. Proteins are sorted
and packaged into vesicles for further transport.

3. Lysosomes: Proteins destined for lysosomes receive a mannose-6-phosphate tag

in the Golgi, directing them to lysosomes.

4. Plasma Membrane/Extracellular Space: Secretory proteins or membrane

proteins are packaged into vesicles and transported to the plasma membrane.
Exocytosis releases proteins into the extracellular environment.

31-Jan-25 48
 Sorting and Translocation in Non-Secretory Pathways
1. Mitochondria:

 Proteins with mitochondrial targeting signals are recognized by translocases of

the outer membrane (TOM) and inner membrane (TIM).

 Chaperones assist in protein unfolding for membrane translocation.

2. Nucleus:

 Proteins with nuclear localization signals (NLS) are transported through nuclear
pores via importin proteins.

3. Peroxisomes:

 Peroxisomal proteins contain peroxisomal targeting signals (PTS1/PTS2) and are

transported by specific receptors..

31-Jan-25 50
iv. Role of Post-Translational Modifications in Sorting and
Translocation

31-Jan-25 52
 iv. Role of Post-Translational Modifications in Sorting and
Translocation
1. Glycosylation:

 is the process by which a carbohydrate is covalently attached to a target

macromolecule, typically proteins and lipid

 Modifies proteins in the ER and Golgi to signal correct folding and sorting.

 Example: Mannose-6-phosphate tags direct proteins to lysosomes.

31-Jan-25 53
 iv. Role of Post-Translational Modifications in Sorting and
Translocation

2. Lipidation:

 is the covalent binding of a lipid group to a peptide chain

 Adds lipid groups (e.g., prenylation, palmitoylation) to anchor proteins to
membranes, assisting in membrane targeting.

31-Jan-25 54
 iv. Role of Post-Translational Modifications in Sorting and
Translocation

3. Phosphorylation:
 is a common posttranslational modification in which a phosphate moiety is
covalently bound to an amino acid.

 Alters protein conformation or creates binding sites for sorting proteins,

influencing trafficking.

 Critical in cell cycle pathways

31-Jan-25 55
 iv. Role of Post-Translational Modifications in Sorting and
Translocation

4. Methylation:
 is a post-translational modification (PTM) where a methyl group (CH₃) is
covalently attached to specific amino acid residues in proteins, usually on lysine
or arginine

 plays a vital role in regulating protein function, stability, localization, and

interaction with other molecule

31-Jan-25 56
 iv. Role of Post-Translational Modifications in Sorting and
Translocation

4. Acetylation:
 is a common post-translational modification in eukaryotes and involves the
addition of an acetyl group to nitrogen via reversible and irreversible processes

 plays a vital role in regulating gene expression, protein stability, and interaction
with other proteins

31-Jan-25 57
 iv. Role of Post-Translational Modifications in Sorting and
Translocation

5. Proteolytic Cleavage:
 is a post-translational modification (PTM) in which specific peptide bonds within
a protein are hydrolyzed by protease enzymes.

 This modification is irreversible and plays a vital role in protein maturation,

activation, degradation, and regulation of cellular functions.

31-Jan-25 58
 Regulation of Gene Expression

Is Gene Regulation Necessary?

 By switching genes off when they are not needed, cells

can prevent resources from being wasted.

 There should be natural selection favoring the ability to

switch genes on and off.

 Complex multicellular organisms are produced by cells

that switch genes on and off during development.

31-Jan-25 59
A typical human cell normally expresses
about 3% to 5% of its genes at any given
time.

Cancer results from genes that do not turn

off properly.

Cancer cells have lost their ability to

regulate mitosis, resulting in uncontrolled
cell division
31-Jan-25 60
Principal levels of gene expression and mechanisms
of regulation
Level of gene expression Regulatable processes involved
Gene preparation Uncoating (viruses)
Synthesis of template genome strand
(viruses)
Modification of chromatin structure
(eukaryotes)
Changes in topological and
conformational properties of DNA
Changes in DNA methylation states
Genomic rearrangements
Gene amplification
31-Jan-25 61
Level of gene expression Regulatable processes
involved
Transcription Promoter usage
Formation of active
transcription complexes
Promoter escape
Elongation vs pausing

Termination vs
antitermination
(prokaryotes)
31-Jan-25 62
Level of gene expression Regulatable processes
involved
RNA processing Capping
(Eukaryotes)
Polyadenylation
Splicing of introns
RNA editing

31-Jan-25 63
Level of gene expression Regulatable processes
involved
RNA export Export of mRNA from
nucleus (eukaryotes)
RNA targeting

31-Jan-25 64
Level of gene expression Regulatable processes involved
Protein synthesis Ribosome binding/initiation of protein
synthesis
Codon usage
Frameshifting
Protein modification Chemical modification of residues
Cleavage of polyproteins
Adoption of quaternary structure
Interaction with regulatory proteins
Protein targeting Targeting to cellular compartments
Secretion
Protein loss and degradation

31-Jan-25 65
 Levels of Regulation
Gene expression is regulated by several different mechanisms:

 Transcriptional control is the most important: binding of proteins to

control regions adjacent to genes that cause RNA polymerase to
transcribe the genes or not.

 Post-transcriptional regulation includes control of RNA: splicing,

transport to specific parts of the cell, stability; and protein: translation,
processing, stability.

 Long range controls: chromatin structure

 Epigenetic mechanisms: control that is inherited during cell division

but which doesn’t involve altering the DNA base sequence.
 Transcriptional Control

• The basic situation is that proteins binding

to DNA sequences near the gene cause or
prevent transcription.

• Proteins that bind to DNA regulatory

sequences and affect transcription are
called transcription factors.
• Transcription factors, which migrate from
the ribosomes to their site of action are
said to act in trans actiing factors.

• In contrast, the DNA regulatory sequences

adjacent to the gene are said to act in cis
acting elements, that they only affect the gene
they are attached to (and not other copies of
the gene in the cell).

• Some transcription factors are general:

involved in all transcription complexes, while
others are specific: only used in certain
tissues or with certain stimuli.

• The latter are often called “tissue-specific 68

transcription factors”.
 Cis-Acting DNA Sequences
 The most important DNA regulatory sequence is the promoter, the
place where RNA polymerase binds and starts transcription.

 The best known is the TATA box, located about 25 bp upstream from
the transcription initiation point.

 Like all these elements, the TATA box is a consensus sequence, and
it is not present in all genes.

 Tissue-specific sequences are usually upstream from the promoter,

and consist of short consensus sequences.
70
 Enhancers and Silencers
 Enhancers and silencers are tissue-specific cis-acting DNA
sequences that increase or decrease transcription
regardless of their position

 Enhancers and silencers work by bending the DNA to help

transcription factors bind to the promoter.
 Epigenetic mechanisms
Epigenetics
 is a scientific branch that examines heritable
changes in the phenotype of an organism or in the gene
expression(not caused by the change in the DNA sequence)

31-Jan-25 72
• DNA carries two forms of information: genetic
information in its nucleotide sequence and
epigenetic information in its structure.

• Methylation is one source of epigenetic

information: it is heritable(due to maintenance
methylation) CpG CH4

31-Jan-25 73
How do epigenetic modifications affect genes?
 Every cell in the body has the same genetic
information.

 What makes cells, tissues and organs different is

that different sets of genes are turned on or off
based how genes can interact with the cell’s
transcribing machinery and epigenetic
modifications

E.g. Cellular differentiation processes rely almost

31-Jan-25 74
on epigenetic mechanisms
 Epigenetic mechanisms of gene regulation
 Involve changes in gene expression that do not alter the
underlying DNA sequence but instead affect how genes are
read and transcribed.

 These modifications can be influenced by environmental

factors and can be passed on during cell division.
 Main Epigenetic mechanisms of gene regulation
1. DNA Methylation:

 Methyl groups are added to the cytosine bases in DNA,

typically at CpG sites.

 This modification usually represses gene expression by

preventing transcription factors from binding to the
promoter region.
 DNA Methylation
 The methylation state of DNA is maintained through
mitosis: daughter cells are methylated in the same way as
the parent cell. Methylation changes are thus epigenetic
changes: heritable changes that don’t alter the DNA base
sequence.

 When DNA replicates, an enzyme called “maintainence

methylase” recognizes methylated cytosines on the old
strand (in a CpG dinucleotide), and methylates the
corresponding C on the new strand.
78
 Main Epigenetic mechanisms of gene regulation

2. Histone Modification:
 Histone proteins, around which
DNA is wrapped, can undergo
various post-translational
modifications (e.g., acetylation,
methylation, phosphorylation).

 These changes can either

condense the chromatin
structure (leading to gene
repression) or relax it (allowing
gene activation).
Covalent histone modifications
 Main Epigenetic mechanisms of gene regulation
Histon Acetylation:
 Acetylation of histones
usually relaxes the
chromatin structure,
making the DNA more
accessible to
transcriptional machinery
and increasing gene
expression.
 Histone Acetylation
 Histones are basic proteins: lysines have a + charge that is attracted to
the – charges on DNA phosphates.

 Histone acetylases add acetate (CH3COOH) to the NH2 at the end of

lysine. This removes the + charge, and in consequence the histones
are less tightly bound to the DNA.
• Genes in the region of acetylated histones are
active; non-acetylated histones are associated
with inactive genes.

• The chromatin in areas of acetylated histones is

less condensed.

• Histone acetylases and de-acetylases can be part

of transcriptional complexes, helping to activate
specific genes.

83
Animation Video
 Applications of Molecular Biology in various
Fields
 Molecular biology is a rapidly advancing field with
applications spanning across diverse areas such as:
 medicine,

 agriculture,

 environmental science,

 industrial processes.
 1. Medicine and Healthcare
 Molecular biology has revolutionized the understanding,
diagnosis, and treatment of diseases.
 A. Genetic Testing and Diagnosis:
 Identifying genetic disorders
(e.g., cystic fibrosis, sickle cell
anemia) through DNA
sequencing.

 Prenatal genetic testing for early

detection of congenital
disorders.

 Personalized medicine:
Tailoring treatments based on
an individual’s genetic makeup.
 B. Gene Therapy:
 is a technique in medical science designed to treat or prevent
diseases by directly modifying or manipulating genes.

 It involves introducing, removing, or altering genetic material within a

patient's cells to correct or compensate for a disease-causing genetic
defect.

 Used in treating genetic disorders like SCID (Severe Combined

Immunodeficiency)
 C. Pharmaceutical Development:
 Designing drugs targeting specific molecular pathways
(e.g., monoclonal antibodies, antivirals).

 Development of vaccines, including mRNA-based

vaccines like those for COVID-19.
 D. Cancer Research:
 Identification of oncogenes and tumor suppressor genes.

 Development of molecular-targeted therapies (e.g., HER2

inhibitors in breast cancer).
 2. Agriculture
 Molecular biology enhances crop production, disease
resistance, and sustainability.
A. Genetically Modified Organisms (GMOs):

 Development of pest-resistant crops (e.g., Bt cotton, Bt

corn).

 Drought-tolerant and salinity-resistant crops (e.g.,

genetically modified rice).

 Fortification (Golden rice)

 B. Synthetic Biology in Agriculture:

 Designing crops with enhanced nutritional content (e.g.,

Golden Rice rich in Vitamin A).
 C. Marker-Assisted Selection:

 Identifying genetic markers linked to desirable traits for

crop improvement.
 3. Environmental Science
 Molecular biology plays a vital role in addressing
environmental challenges by providing tools for
monitoring, remediation, and sustainability.
 A. Bioremediation
 Using genetically engineered
microorganisms (GEMs) to
detoxify or degrade
environmental pollutants.

 Examples:

 Bacteria engineered to clean

up oil spills by breaking down
hydrocarbons.

 Microbes modified to detoxify

heavy metals like mercury
and arsenic in contaminated
sites.
 B. Environmental Monitoring
 Molecular techniques enable the
detection and monitoring of
pollutants, biodiversity, and
ecosystem health.

a) Environmental DNA
(eDNA):Collecting DNA from soil,
water, or air samples to identify
species present in the
environment.

Applications:

 Monitoring endangered species

 Detecting invasive species before

they become established.
 B. Environmental Monitoring
b) Biosensors
 Genetically engineered microorganisms or proteins that act as sensors
for specific pollutants (e.g., nitrates, pesticides, or toxins).
 D. Carbon Sequestration
 Molecular biology techniques are used to enhance carbon capture
processes.

 Examples:

 Genetically engineered algae or cyanobacteria with improved

photosynthetic efficiency to absorb more CO₂
 E. Bioenergy Production
 Molecular biology aids in developing biofuels and reducing reliance on
fossil fuels.

 Examples:

 Engineering algae to produce bioethanol, biodiesel, or biogas.

 Optimizing microbial fermentation pathways for efficient biofuel

production.
 F. Addressing Climate Change
 Molecular tools are used to study the genetic and physiological adaptations of
organisms to climate change

 Examples:

 Understanding how organisms respond to temperature changes and

increasing CO₂ levels.

 Designing stress-tolerant plants or microbes to cope with changing

environments.
 G. Phytoremediation
 Enhancing plants through genetic engineering to absorb and detoxify
pollutants from the soil and water

 Examples:

 Genetically modified plants to absorb heavy metals like lead and cadmium.

 Use of engineered plants to remove excess nitrogen from agricultural runoff.

 H. Detection of Pathogens and Contaminants
 Rapid detection of harmful microorganisms and pathogens in water, soil, and
air using molecular techniques

 Examples:

 PCR-based methods to detect E. coli in water supplies.

 Identifying antibiotic-resistant bacteria in natural ecosystems.

 4. Industrial Processes
 Molecular biology has revolutionized industrial
processes, providing innovative solutions to
global challenges in energy, health, food, and
sustainability.

 By harnessing genetic engineering, recombinant

DNA technology, and synthetic biology, industries
can achieve greater efficiency, reduce
environmental impact, and develop novel
products.
 A. Biotechnology in Fermentation Industries
 Production of Enzymes: Molecular biology enables the
production of industrial enzymes like amylases, lipases,
and proteases, widely used in detergents, food processing,
and biofuel production.
 A. Biotechnology in Fermentation Industries
 Genetic Modification of Microorganisms: Genetic engineering
techniques improve the metabolic pathways of microorganisms,
enhancing fermentation yields for products such as ethanol, citric acid,
and antibiotics.
 A. Biotechnology in Fermentation Industries
 Optimized Fermentation Conditions: Molecular
tools are used to analyze and optimize the genes
involved in fermentation, ensuring higher
efficiency and cost-effectiveness.
 B. Pharmaceutical Industry
 Recombinant Protein Production: Molecular biology techniques allow
for the production of human insulin, growth hormones, and
monoclonal antibodies using genetically modified organisms (e.g.,
bacteria or yeast).

 Vaccine Development: Techniques like recombinant DNA technology

and CRISPR enable the development of safer and more effective
vaccines, such as mRNA vaccines.

 Antibiotic Synthesis: Molecular tools help discover and engineer novel

antibiotics and improve their production processes.
 C. Biofuel Production
 Engineering Microorganisms: Molecular biology
facilitates the engineering of microbes to produce
biofuels like ethanol, butanol, and biodiesel from
renewable biomass sources.

 Enzyme Engineering: Enzymes like cellulases and

hemicellulases are optimized through genetic
modifications to enhance the breakdown of plant
material into fermentable sugars.
 D. Food and Beverage Industry
 Starter Cultures: Molecular techniques help enhance the
quality and consistency of starter cultures used in yogurt,
cheese, and beer production.

 Biosensors: Biosensors based on molecular tools are

employed to detect contaminants and ensure food safety.
 E. Textile and Leather Industry
 Enzymatic Processing: Genetically engineered
enzymes are used for fabric softening, stain
removal, and leather tanning, making the
processes eco-friendly.

 Dye Production: Molecular biology aids in the

biosynthesis of natural dyes, reducing reliance on
harmful chemical dyes.
 F. Production of Bioplastics
 Polymer Synthesis: Bacteria such as Ralstonia
eutropha are genetically engineered to produce
biodegradable plastics like polyhydroxyalkanoates
(PHAs).

 Pathway Optimization: Molecular tools optimize

the metabolic pathways of organisms for large-
scale production of bioplastics.
• RECOMBINANT DNA TECHNOLOGY
(GENETIC ENGINEERING)
 Recombinant DNA is DNA that has been artificially manipulated
to combine genes from two different sources.

 Genes can be transferred among unrelated species via laboratory

manipulation, called Recombinant DNA Technology (genetic
engineering).
 -Genes are cloned by taking a piece of DNA from an
organism and placing it into a cloning vector to
make a recombinant DNA molecule

-The basic procedure of the recombinant DNA

technique consists of two stages
1) Joining a DNA segment, which is of interest for
some reason to a DNA molecule that is able to
replicate,
2) Providing a milieu that allows propagation of the
joined unit

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 VECTOR (carrier molecule or cloning vehicle)
● A vector must have three properties
1) It must be able to replicate
2) There must be some way to introduce vector DNA into a
cell
3) There must be some means of detecting its present,
preferably by a plating test in petri dishes

– E.g. the three most common types of cloning vectors in use

are:
● Plasmids,

● E.coli phage Lambda, λ, and

● Cosmids

1/31/2025 117
● Different types of cloning vectors are used for
different types of cloning experiments.
● The vector is chosen according to the size and type
of DNA to be cloned
● Plasmid vectors are used to
clone DNA ranging in size
from several base pairs to
several thousands of base
pairs (100bp -10kb).
● pUC vehicles commercially
available ones, eg pGEM3,
pBlueScript
● Cannot accept large fragments
● Sizes range from 0- 10 kb
● Standard methods of transformation are
inefficient
● Phage lambda is a bacteriophage or phage, i.e. bacterial
virus, that uses E. coli as host.
● Its structure is that of a typical phage: head, tail, tail fibres.
● Lambda viral genome: 48.5 kb linear DNA with a 12 base
ssDNA "sticky end" at both ends; these ends are
complementary in sequence and can hybridize to each other
(this is the cos site: cohesive ends).
● Infection: lambda tail fibres adsorb to a cell surface receptor,
the tail contracts, and the DNA is injected.
● The DNA circularizes at the cos site, and lambda begins its
life cycle in the E. coli host.
● Purpose:
1. Clone large inserts of
DNA: size ~ 45 kb
● Features:
Cosmids are Plasmids with
one or two Lambda Cos
sites.
● Presence of the Cos site
permits in vitro packaging
of cosmid DNA into
Lambda particles
advantages as cloning vehicle:
● Strong selection for cloning of large inserts
● Infection process rather than transformation for
entry of chimeric DNA into E. coli host
● Maintain Cosmids as phage particles in solution
● But Cosmids are Plasmids:
Thus do NOT form plaques but rather cloning
proceeds via E. coli colony formation
 Bacterial:
 Yeast: expression vectors: plasmids, yeast
artifical chromosomes (YACs)
 Insect cells:
 Mammalian:
● Allows a cloned segment of DNA to be translated
into protein inside a bacterial or eukaryotic cell.
● Bacterial expression vectors typically have two

elements that are required for active gene

expression:
a) a strong promoter
b) a ribosome binding site near an initiating ATG
codon
● Produces large amounts of a specific
protein
● Permits studies of the structure and
function of proteins
● Can be useful when proteins are rare
cellular components or difficult to isolate
● Shuttle vectors can replicate in two different
organisms, e.g. bacteria and yeast, or mammalian
cells and bacteria.
● They have the appropriate origins of replication.
● Hence one can clone a gene in bacteria, maybe
modify it or mutate it in bacteria, and test its
function by introducing it into yeast or animal
cells.
● small size (easy to manipulate and isolate)

● circular (more stable)

● replication independent of host cell

● several copies may be present (facilitates replication)

● frequently have antibiotic resistance (detection easy)

 To open up the DNA a restriction enzyme is used.

 Cut the DNA at a specific place called a restriction site.

 The result is a set of double-stranded DNA pieces with single-

stranded ends

 These ends that jut out are not only "sticky" but they have gaps that
can be now be filled with a piece of foreign DNA

 For DNA from an outside source to bond with an original fragment,

one more enzyme is needed

 DNA ligase seals any breaks in the DNA molecule

 Restriction enzymes: enzymes that
cut DNA in specific places

 Function: Inactivate foreign DNA

 Breaks only palindrome sequences,

i.e. those exhibiting two-fold
symmetry

 Companies purify and market

restriction enzymes
1/31/2025 131
● 5' overhangs: The enzyme cuts asymmetrically within the
recognition site such that a short single-stranded segment extends
from the 5' ends. Bam HI cuts in this manner.
● 3' overhangs: Again, we see asymmetrical cutting within
the recognition site, but the result is a single-stranded
overhang from the two 3' ends. KpnI cuts in this
manner.
● Blunts: Enzymes that cut at precisely opposite sites in the two
strands of DNA generate blunt ends without overhangs. SmaI
is an example of an enzyme that generates blunt ends.
 Cut the cloning vector with R.E. of choice, eg Eco RI

 Cut DNA of interest with same R.E. or R.E. yielding same

sticky ends, e.g. Bam HI and Sau 3A

 Mix the restricted cloning vector and DNA of interest together.

 Ligate fragments together using DNA ligase

 Insert ligated DNA into host of choice - transformation of E.

coli

 Grow host cells under restrictive conditions,

grow on plates containing an antibiotic
 Colony Selection: finding the rare bacterium with
recombinant DNA

 Only E. coli cells with resistant plasmids grow on

antibiotic medium

 Only plasmids with functional lacZ gene can grow

on Xgal
lacZ(+) => blue colonies
lacZ functional => polylinker intact => nothing
inserted, no clone
lacZ(-) => white colonies polylinker disrupted =>
successful insertion & recombination!
● The portion of the lacZ gene encoding the first 146
amino acids (the α -fragment) are on the plasmid

● The remainder of the lacZ gene is found on the

chromosome of the host.

● If the α -fragment of the lacZ gene on the plasmid is

intact (that is, you have a non-recombinant
plasmid), these two fragments of the lacZ gene (one
on the plasmid and the other on the chromosome)
complement each other and will produce a
functional β -galactosidase enzyme.
● In the example shown above, the b-galactosidase gene is
inactivated. The substrate "X-gal" turns blue if the gene
is intact, ie. makes active enzyme. White colonies in X-gal
imply the presence of recombinant DNA in the plasmid.
● Involves five steps:
Enzyme restriction digest of DNA sample.
Enzyme restriction digest of DNA plasmid vector.
Ligation of DNA sample products and plasmid vector.
Transformation with the ligation products.
Growth on agar plates with selection for antibiotic resistance.
● The process of transferring exogenous DNA into
cells is call “transformation”
● There are basically two general methods for
transforming bacteria. The first is a chemical
method utilizing CaCl2 and heat shock to promote
DNA entry into cells.
● A second method is called electroporation based on
a short pulse of electric charge to facilitate DNA
uptake.
● Blue colonies represent Ampicillin-resistant bacteria
that contain pVector and express a functional alpha
fragment from an intact LacZ alpha coding sequence.

White colonies represent Ampicillin-resistant bacteria

that contain pInsert and do not produce LacZ alpha
fragment