3. 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
Genes that code for amino acid sequences are known as
'structural genes'.
Gene expression
4. The process of gene expression involves two main stages:
1. Transcription: the production of messenger RNA (mRNA)
by the enzyme RNA polymerase, and the processing of the
resulting mRNA molecule.
2. Translation: the use of mRNA to direct protein synthesis,
and the subsequent post-translational processing of the protein
molecule.
5. Transcription involves four steps:
1. Initiation. The DNA molecule unwinds and separates to
form a small open complex. RNA polymerase binds to the
promoter of the template strand
2. Elongation. RNA polymerase moves along the template
strand, synthesising an mRNA molecule. In prokaryotes RNA
polymerase is a holoenzyme consisting of a number of
subunits, including a sigma factor (transcription factor) that
recognises the promoter. In eukaryotes there are three RNA
polymerases: I, II and III. The process includes a
proofreading mechanism.
Transcription
6. Mammalian Cells Possess Three Distinct Nuclear
DNA-Dependent RNA Polymerases
Form of RNA
polymerase
Major products
I rRNA
II mRNA, miRNA
III tRNA/5S rRNA,
snRNA
Transcription Contd...
7. 3. Termination. In prokaryotes there are two ways in
which transcription is terminated. In Rho-dependent
termination, a protein factor called "Rho" is responsible
for disrupting the complex involving the template strand,
RNA polymerase and RNA molecule. In Rho-independent
termination, a loop forms at the end of the RNA molecule,
causing it to detach itself. Termination in eukaryotes is
more complicated, involving the addition of additional
adenine nucleotides at the 3' of the RNA transcript (a
process referred to as polyadenylation).
Transcription Contd...
8. 4. Processing. After transcription the RNA molecule is
processed in a number of ways: introns are removed and the
exons are spliced together to form a mature mRNA molecule
consisting of a single protein-coding sequence. RNA synthesis
involves the normal base pairing rules, but the base thymine is
replaced with the base uracil.
9. Translation involves four steps:
1. Initiation. The small subunit of the ribosome binds at the
5' end of the mRNA molecule and moves in a 3' direction until
it meets a start codon (AUG). It then forms a complex with the
large unit of the ribosome complex and an initiation tRNA
Molecule
2. Elongation. Subsequent codons on the mRNA molecule
determine which tRNA molecule linked to an amino acid binds
to the mRNA. An enzyme peptidyl transferase links the amino
acids together using peptide bonds. The process continues,
producing a chain of amino acids as the ribosome moves along
the mRNA molecule.
10. 3. Termination. Translation in terminated when the ribosomal
complex reached one or more stop codons (UAA, UAG, UGA).
The ribosomal complex in eukaryotes is larger and more
complicated than in prokaryotes. In addition, the processes of
transcription and translation are divided in eukaryotes
between the nucleus (transcription) and the cytoplasm
(translation), which provides more opportunities for the
regulation of gene expression.
4. Post-translation processing of the protein
11. 1. Exons.
• Exons code for amino acids and collectively
determine the amino acid sequence of the protein product
•It is these portions of the gene that are represented in final
mature mRNA molecule.
2. Introns.
•Introns are portions of the gene that do not code
for amino acids, and are removed (spliced) from the mRNA
molecule before translation.
A structural gene involves a number of different components:
12. Gene regulation is a label for the cellular processes that
control the rate and manner of gene expression
A complex set of interactions between genes, RNA molecules,
proteins (including transcription factors) and other
components of the expression system determine
i. when and where specific genes are activated and
i. the amount of protein or RNA product produced
Gene regulation
13. Some genes are expressed continuously, as they produce
proteins involved in basic metabolic functions;
Some genes are expressed as part of the process of cell
differentiation;
and some genes are expressed as a result of cell
differentiation.
Gene regulation
14. Mechanisms of gene regulation include:
Regulating the rate of transcription
This is the most economical method of regulation
Regulating the processing of RNA molecules, including
alternative splicing to produce more than one protein product
from a single gene
Regulating the stability of mRNA molecules
Regulating the rate of translation
Epigenetic factors
15. The regulatory sequence of a cloned eukaryotic gene is ligated to a
reporter gene that encodes an easily detectable enzyme. The
resulting plasmid is then introduced into cultured recipient cells
by transfection. An active regulatory sequence directs transcription of the
reporter gene, expression of which is then detected in the transfected
cells.
Identification of eukaryotic regulatory sequences
Gene expression regulatory (sequence) regions
16. Gene expression regulatory (sequence) regions
These include:
1. Transcription start site
2.Promoter
3.Enhancers
4.Silencers
5.Insulator
17. Start site-
is where transcription of the gene into RNA begins
This is where a molecule of RNA polymerase II binds
TGTTGACS TATAAT
Termination
signal
Coding
strand
Template
strand
5’
3’
Transcrition
Start site
+1
PPP
5’
OH
3’
RNA
3’
5’
DNA
-35
region
-10
region
5’ flanking
sequences
3’ flanking
sequences
Promoter Transcribed
region
Gene expression regulatory (sequence) regions
18. A promoter-
Initiates transcription. It is not transcribed into
mRNA, but plays a role in controlling the transcription of the
Gene
Cis-acting sequence
1. Basal or core promoter - A region located within about 40 bp
of the start size
2. Upstream promoter- a region which may extend over as
many as few hundred nucleotides further 'upstream' of the
gene (toward the 5' end).
Gene expression regulatory (sequence) regions
19. Genes transcribed by RNA polymerase II have two core
promoter elements,
1. the TATA box and
2. the Inr sequence,
that serve as specific binding sites for general transcription
Factors
The TATA box is bound by a large complex of some 50
different proteins, including:
I. Transcription factor IID (TFIID), which is a complex of
a. TATA-binding protein (TBP), which recognizes an binds to
the TATA box
20. b. 13 other protein factors, which bind to TBP, each other and
(some of them) to the DNA
II. Transcription factor IIB (TFIIB), which binds both the
DNA and pol II
TGTTGACS TATAAT
Termination
signal
Coding
strand
Template
strand
5’
3’
Transcrition
Start site
+1
PPP
5’
OH
3’
RNA
3’
5’
DNA
-35
region
-10
region
5’ flanking
sequences
3’ flanking
sequences
Promoter Transcribed
region
21. Enhancers-
Some transcription factors (called activators or enhancer
binding protein, EBP) bind to regions called 'enhancers' that
increase the rate of transcription. These sequences stimulate
transcription from promoter
Their activity depend on neither their distance nor their
orientation with respect to the transcription initiation site
These sites may be thousands of nucleotides from the
coding sequences or within an intron. Some enhancers are
conditional and only work in the presence of other factors as
well as transcription factors
Gene expression regulatory (sequence) regions
22. Action of enhancers
Without an enhancer, the
gene is transcribed at a low
basal level (A).
Addition of an enhancer,
E—for example, the SV40
72-base-pair repeats—
stimulates transcription.
The enhancer is active not
only when placed just
upstream of the promoter
(B)
23. but also when inserted up to
several kilobases either
upstream or downstream
from the transcription start
site (C and D)
In addition, enhancers are
active in either the forward
or backward orientation (E)
24.
25. The ability of enhancers to function even when separated by
long distances from transcription initiation sites at first
suggested that they work by mechanisms different from those
of promoters
However, this has turned out not to be the case: Enhancers,
like promoters, function by binding transcription factors that
then regulate RNA polymerase
Gene expression regulatory (sequence) regions
26. Gene expression regulatory (sequence) regions
This is possible because
of DNA looping, which allows
a transcription factor bound
to a distant enhancer to
interact with RNA
polymerase or general
transcription factors at the
promoter
27. DNA looping
Transcription factors bound at
distant enhancers (enhancer
binding proteins, EBP) are able
to interact with general
transcription factors at
the promoter
because the
intervening DNA can form loops.
There is therefore no
fundamental difference between
the action of transcription
factors bound to DNA just
upstream of the promoter and
to distant enhancers.
Gene expression regulatory (sequence) regions
28. Silencers-
Some transcription factors (called repressors) bind to
regions called 'silencers' that depress the rate of
transcription
Gene expression regulatory (sequence) regions
29. Insulator-
Stretches of DNA located between the
I. Enhancer(s) and promoter(s) or
II. Silencer(s) and promoter(s)
of adjacent genes or clusters of adjacent genes
Their function is to prevent a gene from being influenced
by the activation (or repression) of its neighbor
Gene expression regulatory (sequence) regions
insulator
30. The isolation of a variety of transcriptional regulatory
proteins has been based on their specific binding to promoter
or enhancer sequences
Protein binding to these DNA sequences is commonly analyzed
by two types of experiments
1. The first, footprinting,
2. The second approach is the electrophoretic-mobility shift
assay, in which a radiolabeled DNA fragment is incubated with
a protein preparation and then subjected to electrophoresis
through a nondenaturing gel sites of specific DNA-binding
proteins
Transcriptional regulatory proteins
31. Protein binding is detected as a decrease in the
electrophoretic mobility of the DNA fragment, since its
migration through the gel is slowed by the bound protein
The combined use of footprinting and electrophoretic-
mobility shift assays has led to the correlation of
protein-binding sites with the regulatory elements of
enhancers and promoters, indicating that these sequences
generally constitute the recognition
32. Footprinting
A technique derived from principles used in DNA sequencing,
identifies the DNA sequences bound by a particular protein
DNA frament thought to contain sequences recognised by a
DNA-binding protein and radiolabelled one end of one strand
Chemical or enzymatic reagents break the DNA into fragments
Randomly
Seperation of the labelled cleavage products (broken
Fragments of various lengths) by high resolution electrophoresis
Produces a ladder of radioactive bands
In a separate tube, the cleavage procedure is repeated on
Copies of the same DNA fragment in the presence of the DNA-
Binding protein
33. The two sets of cleavage products are subjected to
electrophoresis, and compare them side by side
A gap (“footprint”) in the series of radioactive bands derived
from the DNA-protein sample attribute to protection of the
DNA by the bound protein
Identifies the sequences that the protein bound
The precise location of the protein-binding site can be
fetermined by directly sequencing copies of the same DNA
Fragment and including the sequencing lanes on the same gel
Footprinting
35. A sample containing radiolabeled fragments of DNA is divided
into two, and one half of the sample is incubated with a protein
that binds to a specific DNA sequence. Samples are then
analyzed by electrophoresis in a nondenaturing gel so that the
protein remains bound to DNA.
Electrophoretic-mobility shift assay *EMSA)
36. Protein binding is detected by the slower migration of
DNA-protein complexes compared to that of free DNA. Only a
fraction of the DNA in the sample is actually bound to protein,
so both DNA protein complexes and free DNA are detected
following incubation of the DNA with protein.
Electrophoretic-mobility shift assay (EMSA)
37. Transcriptional regulatory proteins
These include:
1. General transcription factor
2.Transcriptional activators
3.Transcriptionl repressors
https://www.ncbi.nlm.nih.gov/books/NBK9904/
38. Transcription factors-
are proteins that play a role in regulating the transcription of
genes by binding to specific regulatory nucleotide sequences
Transcription factors bind to specific nucleotide sequences
in the promoter region and assist in the binding of
RNA polymerases
Transcriptional regulatory proteins
39. Transcription factor Consensus binding site
Specificity protein 1 (Sp1) GGGCGG
CCAAT/Enhancer binding
protein (C/EBP)
CCAAT
Activator protein 1 (AP1) TGACTCA
Octamer binding proteins ATGCAAAT
(OCT-1 and OCT-2)
E-box
binding proteins (E12, E47,
E2-2)
CANNTG
a
Examples of Transcription Factors and Their DNA-Binding
Sites
a = N stands for any nucleotide.
40. Transcriptional activator
The most thoroughly studied of
these proteins are transcriptional activators, which, like Sp1,
bind to regulatory DNA sequences and stimulate transcription
In general, these factors have
been found to consist of two
domains:
1. One region of the protein
specifically binds DNA
2. the other activates
transcription by interacting
with other components of
the transcriptional machinery
41. Transcriptional activators appear to be modular
proteins, in the sense that the DNA binding and
activation domains of different factors can frequently
be interchanged using recombinant DNA techniques
Such manipulations result in hybrid transcription
factors, which activate transcription by binding
to promoter or enhancer sequences determined by the
specificity of their DNA-binding domains
It therefore appears that the basic function of the
DNA-binding domain is to anchor the transcription
factor to the proper site on DNA; the activation domain
then independently stimulates transcription by
interacting with other proteins.
42. Structure of transcriptional activators
Transcriptional activators consist of two
independent domains
1. The DNA-binding domain recognizes a specific DNA
sequence, and
2. the activation domain interacts with other components
of the transcriptional machinery.
43. DNA-binding domain
Molecular characterization has revealed that the DNA-
binding domains of many of these proteins are related to one
another . These include:
1. Zinc-finger domain
2. Helix-turn-helix motif
3. Leucine zipper
4. Helix loop-helix
44. 1. Zinc finger domains
contain repeats of cysteine and histidine residues that
bind zinc ions and fold into looped structures (“fingers”)
that bind DNA
These domains were initially identified in the polymerase
III transcription factor (TFIIIA) but are also common
among transcription factors that regulate polymerase II
promoters, including Sp1
Other examples of transcription factors that contain
zinc finger domains are the steroid hormone receptors,
which regulate gene transcription in response
to hormones such as estrogen and testosterone
DNA-binding domain
45. 2. The helix-turn-helix motif
was first recognized in
prokaryotic DNA
binding proteins, including
the E. coli catabolite activator
protein (CAP)
In these proteins, one helix
makes most of the contacts with
DNA, while the other helices lie
across the complex to stabilize
the interaction
In eukaryotic cells, helix-turn-
helix proteins include
the homeodomain proteins, which
play critical roles in the
regulation of gene expression
46. leucine zipper and helix-loop-
helix proteins, contain DNA-
binding domains formed by
dimerization of
two polypeptide chains
3. The leucine zipper
contains four or five leucine
residues spaced at intervals of
seven amino acids, resulting in
their hydrophobic side chains being
exposed at one side of a helical
region
This region serves as the
dimerization domain for the two
protein subunits, which are held
together by hydrophobic
interactions between the leucine
side chains.
47. Immediately following the leucine zipper is a region rich
in positively charged amino acids (lysine and arginine) that
binds DNA.
48. 4. The helix-loop-helix
proteins are similar in
structure to the leucine
zipper, except that their
dimerization domains are each
formed by two helical regions
separated by a loop
49. An important feature of both leucine zipper and
helix-loop-helix transcription factors is that different
members of these families can dimerize with each other
Both leucine zipper and helix-loop-helix proteins play
important roles in regulating tissue-specific and
inducible gene expression, and the formation of dimers
between different members of these families is a critical
aspect of the control of their function
50. Activation domain
Are not as well characterized as their DNA-binding domains
acidic activation domains, are rich in negatively charged
residues (aspartate and glutamate); others are rich in proline
or glutamine residues
These activation domains are thought to stimulate
transcription by interacting with general transcription factors,
such as TFIIB or TFIID, thereby facilitating the assembly of
a transcription complex on the promoter
51. For example, the activation
domains of several transcription
factors (including Sp1) have
been shown to interact with
TFIID by binding to TBP-
associated factors (TAFs)
An important feature of these
interactions is that different
activators can bind to different
general transcription factors or
TAFs, providing a mechanism by
which the combined action of
multiple factors can
synergistically stimulate
transcription—a key feature of
transcriptional regulation
in eukaryotic cells
52. Gene expression in eukaryotic cells is regulated by repressors
as well as by transcriptional activators
Like their prokaryotic counterparts, eukaryotic repressors
bind to specific DNA sequences and inhibit transcription
In some cases, eukaryotic repressors simply interfere with the
binding of other transcription factors to DNA
.
Eukaryotic repressors
53. For example, the binding of a repressor near the transcription
start site can
1. block the interaction of RNA polymerase or general
transcription factors with the promoter, which is similar to the
action of repressors in bacteria
2. Other repressors compete with activators for binding
to specific regulatory sequences. Some such repressors contain
the same DNA-binding domain as the activator but lack its
activation domain. As a result, their binding to a
promoter or enhancer blocks the binding of the activator,
thereby inhibiting transcription
54. (A)Some repressors block the binding of activators to
regulatory sequences.
(B)Other repressors have active repression domains that
inhibit transcription by interactions with general
transcription factors
Action of eukaryotic repressors
55. Post transcription regulation of gene
expression
Primary transcript in nucleus most undergo processing steps
to produce functional RNA molecules for export to cytosol
Steps of RNA processing:
1. Synthesis of the ‘cap’’
A modified guanine (G) attached at the 5’ end of the pre-
mRNA (primary transcript)
Cap protects the RNA from enzyme degradation
Serves as an assembly point for proteins needed to recruit
The small subunit of the ribosome
2. Step by step removal of introns present in the pre-mRNA
and splicing
3. Synthesis of the poly(A) tail. This is a stretch of adenine
(A) nucleotides
56. Removal of introns and splicing of exons is done by splicosomes
These are a complexes of 5 snRNA molecules and some 145
different proteins
Post transcription regulation of gene
expression
57. Epigenetic factors in gene regulation-
chromatin structure in relation to
transription regulation
Eukaryotic genes are not naked DNA within the nucleus
DNA in eukaryotic cells is tightly bound to histones forming
Chromatins
The basic structural unit of chromatin is the nucleosome,
consisting of 146 bp of DNA wrapped around 2 molecules each
of histones H2A, H2B, H3 and H4 with one molecule of H1
bound to the DNA as it enters the nucleosomes core particle
Chromatin: DNA + Histones (basic proteins) + nonhistone
proteins + RNA
58. Nucleosomes are composed of DNA wound around a
collection of histone molecules
Histone octamer
DNA
Histone
H1
Nucleosome structure
The super-packing of nucleosomes in nuclei is seemingly
dependent upon the interaction of the H1 histones with
adjacent nucleosomes
59. This packaging of eukaryotic DNA in chromatin has important
consequences in terms of its availability as a template for
Transcription
So chromatin structure is a critical aspect of gene expression
In eukaryotic cells
Actively transcribed genes are found in decondensed
Chromatin
Decondensation of chromatin is not sufficient to make the
DNA an accessible template for transcription
Actively transcribed genes remain bound to histones and
packaged in nucleosomes
60. So transcription factors and RNA polymerase are still faced
with the problem of interacting with chromatin rather than
with naked DNA
The tight winding of DNA around the nucleus core particle is a
major obstacle for transcription
This inhibitory effect of nucleosomes is relieved by:
1. acetylation of histones and
2. the binding of two nonhistone chromosomal proteins (called
HMG-14 and HMG-17) to nucleosomes of actively transcribed
genes
3. nucleosome remodelling factor-a protein that facilitate the
binding of transcription factors to chromatin by altering
nucleosome structure
HMG-High mobility group proteins
61. Acetylation
Core histones- H2A, H2B, H3 and H4 have 2 domains:
a. A histone fold domain- involved in interactions with other
histones and in wrapping DNA around the nucleosome core
particle
b. An amino-terminal tail domain- extends outside of the
nucleosome
Amino-terminal tail domains are rich in lysine and can be
modified by acetylation at specific lysine residues
Acetylation reduces the net positive charge of the histones
and may weaken their binding to DNA and their interactions
with other proteins
Acetylation facilitates binding of TF to nucleosomal DNA
Transcriptional activators are associated with
acetyltransferases
Transcriptional repressors are associated with deacetylase
62. Nonhistone chromosomal proteins (HMG-17 &
HMG-14)
They facilitate the ability of RNA polymerase to transcribe
chromatin template
They alter the interaction of H1 with nucleosomes to maintain
a decondensed chromatin structure that facilitate
transcription through a nucleosome template. Because their
binding site on nucleosome overlap that of H1
Histone octamer
DNA
Histone
H1
63. Nucleosome remodelling factors
Are protein complexes that facilitate the binding of TFs by
altering nucleosome structure
Mechanism not yet clear, but increase the accessibility of
nucleosomal DNA to other proteins (e.g TFs) without removing
the histones
They do this probably by catalysing the sliding of histone
octamers along the DNA molecule, thereby repositioning
nucleosome to facilitate TF binding
Histone octamer
DNA
Histone
H1
64. Features of Prokaryotic Gene Expression
1.Operon
The genes involved in a metabolic pathway in prokaryotes are
often present in a linear array called ‘Operon’ e.g lac operon
An operon can be regulated by a single promoter or
regulatory region
2. Cistron
The cistron is the smallest unit of genetic expression or is
the genetic unit coding for the structure of the subunit of a
protein molecule.
65. 3. Polycistronic mRNA
This is a single mRNA that encodes more than one
separately translated protein
For example, polycistronic lac operon mRNA is translated
into three separate proteins
Operons and polycistyronic mRNAs are common in
bacteria but not in eukaryotes
66. 4. Inducible gene
An inducible gene is one whose expression increases in
response to an inducer or activator
It has low basal rates of transcription
5. Constitutive gene
A gene that is constitutively expressed is genes that are
expressed at a reasonably constant rate and not known to be
subject to regulation
These are often referred to as housekeeping gene
6. Constitutive mutation
A mutation resulting in constitutive expression of a formerly
regulated gene is called a constitutive mutation.
67. The lac Operon- an inducible system
lac Operon is the first control system for enzyme
production worked out at the molecular level and described
the control of enzymes that are produced in response to the
presence of sugar lactose in E. coli cell
This system was described by Jacob and Monod (1961)
They described the molecular mechanisms responsible for
the regulation of the genes involved in the metabolism of
lactose
Glucose and Galactose are produced from lactose by the
action of β-galactosidase
Several proteins involved in lactose metabolism in the E.
coli cell
68. These include:
1. β-galactosidase-converts lactose into glucose and
galactose
2. β-galactoside permease- transport lactose into the cell
3. β-galactoside transacetylase-function unknown.
All of the genes involved in controlling this pathway are
located next to each other on the E.coli chromosome.
Together they form an operon
lacI lacZ lacY lacA
Operator
Promoter
site
lac operon
69. β-Galactosidase hydrolyzes the β-galactoside lactose
to galactose and glucose
The structural gene for β-galactosidase (lacZ) is
clustered with the genes responsible for the permeation
of galactose into the cell (lacY) and for thiogalactoside
transacetylase (lacA)
The structural genes for these three enzymes, along
with the lac promoter and lac operator (a regulatory
region), are physically associated to constitute the lac
operon
lacI lacZ lacY lacA
Operator
Promoter
site
lac operon
70. This genetic arrangement of the structural genes and
their regulatory genes allows for coordinate expression of
the three enzymes concerned with lactose metabolism
Each of these linked genes is transcribed into one large
mRNA molecule that contains multiple independent
translation start (AUG) and stop (UAA) codons for each
cistron
Thus, each protein is translated separately, and they are
not processed from a single large precursor protein. This
type of mRNA molecule is called a polycistronic mRNA
71. Structural and regulatory genes of the lac operon
lacI lacZ lacY lacA
Operator
Promoter
site
lac operon
