Molecular Biology

Dr. Muhammad Saleem

Course Content:
• Regulation of Gene Expression :
1) Transcriptional level:-
Cis Acting Regulatory Factors
2) Translational level:-
• Translation in Eukaryotes.
• Post-translational Modifications.
• Gene rearrangement and Amplification.
cis-Acting Regulatory
Elements(CREs).
Dr. Muhammad Saleem
Protein Engineering Lab, School of Biological
Sciences, University of the Punjab, Lahore.

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Introduction
 cis-acting;

A genetic element, such as an enhancer, a promoter, or

an operator, that must be on the

same chromosome in order to influence a gene’s
activity.

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CREs are short DNA sequences found near genes, often in promoters
or enhancers, that control the timing, location, and level of gene
expression. They are recognized by transcription factors (proteins)
which can either activate or repress the transcription process.

cis-acting regulatory elements

(CREs)
DNA sequences found on the same molecule of DNA as
the genes they regulate.
 CREs involved in regulation of gene expression,
(when and where genes are turned on or off in the genome).
CREs are essential for the precise and coordinated control
of various cellular processes and developmental programs.

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 • In multicellular organisms, gene regulation enables different cell
types (e.g., muscle vs. nerve) to form from the same DNA.
• Proper regulation ensures normal growth and function.
• Cells need to adapt to their environment by turning certain genes
on or off.
Example: Bacteria like E. coli only produce enzymes to digest
lactose when lactose is present.

Why gene regulation is

required

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 cis-acting regulatory elements (CREs)
 Some common cis-acting regulatory elements.

1. Promoters Initiate Transcription

2. Enhancers

3. Silencers Regulate Transcription

4. Insulators

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Location: Can be far from the gene (from 10³ to 10⁶ These are proteins
base pairs away). (not DNA) produced
Function: Enhancers help boost transcription by elsewhere in the
looping the DNA so that proteins bound here (like genome that bind to
transcription factors) can interact with the promoter cis elements (like
region. promoters or
enhancers).
Function: They help
activate or repress
Mediator gene transcription.
Complex
Function: Acts as
a bridge between
transcription
factors at the
enhancer and
RNA polymerase
at the promoter.
Helps assemble
the transcription Location: Just upstream (close) to the gene.
machinery at the Function: Contains core sequences (like the
gene's start site. TATA box) where RNA polymerase II and
transcription factors bind to initiate
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transcription.
eRNA (Enhancer RNA)
Definition: Short RNA molecules transcribed from enhancer regions.
Possible role: May help stabilize enhancer-promoter looping or recruit transcription machinery.
Promoters vs Other Regulatory
Elements
I. The promoter is the DNA sequence where the general
transcription factors and the polymerase assemble.
II. The regulatory sequences serve as binding sites for gene
regulatory proteins, whose presence on the DNA affects the
rate of transcription initiation.
Location; adjacent to the promoter, far upstream of it, or
even within introns or downstream of the gene

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 Promoters

• Specific DNA sequences located near the transcription start

site (TSS) of a gene. They serve as binding sites for RNA
polymerase and transcription factors, initiating the
transcription of DNA into RNA.

• Promoters are critical for regulating when, where, and how

much a gene is expressed.

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 Key Features of Promoters
1. Location:
1. Typically found upstream (before) the gene's coding region.
2. Some promoters are bidirectional (controlling two genes in opposite directions).

2. Core Promoter: It's the minimal sequence required to initiate transcription.

1. Contains essential motifs recognized by RNA polymerase and basal transcription

machinery:
[Link] box: A conserved "TATA" sequence (~25–30 base pairs upstream of
the TSS) that helps position RNA polymerase II.
[Link] (Inr): Surrounds the TSS and aids in accurate transcription initiation.
[Link] Promoter Element (DPE): Found in some genes lacking a TATA box.
for initiation of transcription.
3. Proximal Promoter Region:
1. Located ~250 base pairs upstream of the TSS.
2. Contains binding sites for transcription factors (e.g., Sp1, NF-κB) that enhance or
repress transcription.
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Spacer DNA refers to the non-coding DNA sequences that lie between genes or
regulatory elements.
Maintains the correct distance between important regulatory elements & they can
play important roles in the organization, regulation, and expression of genes.
Promoters vs. Other Regulatory
Elements

The gene control region for a typical eukaryotic

gene.
Alberts (Molecular Biology of the Cell)

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 Types of Promoters
1. Constitutive Promoters:
[Link] active, driving constant gene expression (e.g.,
housekeeping genes like GAPDH or ACTB).

2. Regulated Promoters:
[Link] or repressed by specific signals (e.g., hormones,
stress, development).
[Link]: The lac operon promoter in bacteria responds to
lactose availability.

3. Inducible/Tissue-Specific Promoters:
[Link] only in certain tissues or conditions (e.g., the albumin
promoter in liver cells).
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 Function of Promoters

1. Transcription Initiation:
[Link] RNA polymerase and transcription factors to form the pre-
initiation complex.
[Link] the directionality of transcription.
2. Regulation of Gene Expression:
[Link] of promoter sequences influences transcription rates
(weak vs. strong promoters).
[Link] with enhancers or silencers (distal regulatory
elements) fine- tune expression.
3. Epigenetic Control:
[Link] methylation (e.g., at CpG islands) or histone
modifications (e.g., acetylation) can silence or activate
promoters.
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 Classification of
Promoters
 Three eukaryotic RNA polymerases have different structures

There are 3 different promoters which are recognized by these

polymerases.

 Class I, II and III Promoters

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In eukaryotes, RNA polymerase I (Pol I) specifically transcribes ribosomal RNA (rRNA)
precursor genes — typically the 45S pre-rRNA, which is later processed into 18S, 5.8S, and
28S rRNA.

i. Class I
Promoters
 Class I Promoters are recognized by RNA polymerase I

 All species have only one kind of gene recognized by polymerase I:

the rRNA precursor gene
Exception is the trypanosome, in which polymerase I transcribes two
protein-encoding genes
[Link] (Variant Surface Glycoprotein) genes – important for immune evasion.
[Link] genes – expressed in the insect (tsetse fly) stage)

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 i. Class I
Promoters
Robert Tjian and colleagues identified the important regions of
the human class I promoter.
I. Core element; also known as the initiator (rINR), is located at
the start of transcription, between positions -45 and +20.

II. Upstream promoter element (UPE), located between

positions -156 and -107.
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 i. Class I
Promoters

Two rRNA promoter elements

Weaver (Molecular Biology)

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 ii. Class II
 Promoters
The class II promoters recognized by RNA polymerase II (the
enzyme responsible for transcribing protein-coding genes in eukaryotes)

 These are the most complex and best studied.

 Parts

a. Core promoter

b. The proximal promoter (sometimes called upstream promoter

elements)
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ii. Class II
Promoters
a. Core promoter
• Attracts general transcription factors and RNA polymerase.
• Sets the transcription start site and direction of transcription
• Consists of elements lying within about 37 bp of the
transcription start site
• Core promoter is modular and can contain almost any
combination of the following elements.(continue….)

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ii. Class II
Promoters
a. Core promoter elements
I. The TATA box is centered at approximately position -28
(about -31 to -25); consensus sequence TATA(A/T)AA(G/A)
II. The TFIIB recognition element (BRE) lies just upstream of
the TATA box. Consists of elements lying within about -37 to -
32 bp of the transcription start site

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 ii. Class II
Promoters
III. Initiator (Inr) is centered on the transcription start site (position -
6 to +45), consensus sequence PyPyAN(T/A)PyPy in mammals

[Link] core element (DCE) has three parts located at

approximately +6 to +12, +17 to +23, and +31 to +33,
(consensus sequences CTTC, CTGT, and AGC, respectively)

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 ii. Class II
Promoters
V. Motif ten element (MTE) positions +18 and +27.
VI. Downstream promoter element (DPE) positions +28 and +33.
Note; at least one of these elements is missing in most promoters

A generic class II core promoter.

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 ii. Class II
Promoters
Core promoter elements Functions

Helps TFIIB to bind to

the DNA

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ii. Class II
Promoters

Locate the start of

transcription

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ii. Class II
Promoters
Initiator together
with the TATA box constitutes
core promoter

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ii. Class II
Promoters
In TATA-less promoters,
they compensate function

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 ii. Class II
Promoters
Drive transcription

Weaver (Molecular Biology)

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ii. Class II
Promoters

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ii. Class II
Promoters
b. Proximal promoter elements (UPE)
 Are usually found upstream of class II core promoters

I. GC boxes (GGGCGG & CCGCCC).

(bind the transcription factor Sp1 and stimulates transcription)
II. Cat box (CCAAT)
(binds to CCAAT-binding transcription factor [CTF] to stimulate
transcription)

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 iii. Class III
Promoters
RNA polymerase III transcribes a variety of genes that encode
small RNAs, which includes,
I. The “classical” class III genes (types I and II), including the 5S
rRNA and tRNA genes, and the adenovirus VA RNA genes.
II. Some relatively recently discovered class III genes, including the
U6 snRNA gene, the 7SL RNA gene, the 7SK RNA gene, and
the Epstein–Barr virus EBER2 gene (non-classical)

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 iii. Class III
Promoters
 Classical” class III genes have promoters located internally.
Whereas the promoters recognized by polymerases I and II, as well
as by bacterial polymerases, are located mostly in the 5’-flanking
region.

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 Class III promoters are recognized by RNA Polymerase III (Pol III), which

transcribes small, non-coding RNAs,such as tRNA and 5S rRNA genes,

These promoters have unique internal structures, often located within the gene
itself,— these are known as internal promoters.

Promoter Transcription
Type Example Genes Key Elements
Location Factors

TFIIIA, TFIIIC,
Type I Internal 5S rRNA Box A, IE, Box
C TFIIIB
Type II Internal tRNA Box A, Box B TFIIIC, TFIIIB
External DSE, PSE, TATA SNAPc, TFIIIB
U6 snRNA, 7SK RNA
Type III
(upstream) box (with TBP)

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iii. Class III
Promoters

Promoters of some class III genes.

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 How Transcription Starts from Internal Promoters
1. Promoter elements (Box A and Box B) are embedded within the coding sequence of the gene.

1. Box A is located near the 5' end.

2. Box B is further downstream, still inside the gene body.

2. TFIIIC, a large transcription factor, binds first to Box A and Box B.

1. It acts as a molecular ruler, measuring the spacing between the boxes to ensure correct positioning.

[Link] then recruits TFIIIB, which includes the TBP (TATA-binding protein) even though there's
no TATA box here.

1. TFIIIB binds upstream of the transcription start site (outside the gene body).

4. RNA polymerase III is recruited to the transcription start site by TFIIIB.

5. Transcription initiates just upstream of the gene, despite the promoter being internal.

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 2.
Enhancers
 Enhancers; are cis-acting DNA elements that stimulate
transcription

 Play a critical role in the regulation of gene expression by increasing

the transcription levels of target genes.

 They are non-coding DNA sequences that serve as binding sites

for transcription factors (TFs) and co-activators.

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2.
Enhancers
Enhancers + General transcription factors

Preinitiation complex

Required for Transcription

 Enhancers frequently found upstream of the promoters,

sometimes can be found within a gene.

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2. Enhancers
(Example)
 Different from promoters as they are Orientation- and
Position independent DNA elements.

Structure of the SV40 virus early

control region (Enhancer 72-bp repeats,
blue).
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 3.
AreSilencers
cis-regulatory DNA sequences that function to repress transcription of
target genes. They do so by inhibiting the assembly or function of the
transcriptional machinery, effectively turning genes off or down.

 They are also Orientation- and Position independent DNA elements

They are also tissue-specific in that they rely on tissue-specific DNA-

binding proteins for their activities

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 3.
Silencers
1. Repressor Binding:
[Link] contain binding sites for transcriptional repressors.
[Link] repressors may block transcription factor binding, disrupt
enhancer function, or recruit chromatin modifiers.

2. Chromatin Modification:
[Link] often recruit histone deacetylases (HDACs),
histone methyltransferases, or co-repressor complexes.
[Link] leads to heterochromatin formation, which is
transcriptionally silent.

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3.
Silencers
3. Competition or Interference:
Silencers can interfere with activators by blocking access to
the
promoter or by altering DNA looping dynamics
between enhancers and promoters.

4. Looping to Promoters:
Similar to enhancers, silencers can loop to promoters
to suppress transcription initiation directly.

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3.
Silencers

Mechanism of Heterochromatin formation which causes gene silencing

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Note:
Sometimes the same DNA element can have both enhancer and
silencer activity, depending on the protein bound to it. For
example, the thyroid hormone response element acts as a silencer
when the thyroid hormone receptor binds to it without its ligand,
thyroid hormone. But it acts as an enhancer when the thyroid
hormone receptor binds along with thyroid hormone

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 4.
Insulators
 Are specialized cis-regulatory DNA elements that act as genomic boundary markers.
Their main role is to regulate interactions between enhancers, promoters, and chromatin
domains, ensuring genes are expressed in the right place, at the right time, and at the
right level.

 Are DNA sequences that prevent eukaryotic gene regulatory proteins from influence of
distant genes

 (or) Insulators are DNA elements that can shield genes from activation by enhancers
(enhancer blocking activity) or repression by silencers (barrier activity).

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 Prevent an Stop the spread of
enhancer from heterochromatin into
inappropriately 2. Barrier Activity active gene regions,
1. Enhancer-
activating a protecting genes from
Blocking
nearby promoter transcriptional silencing.
4. if placed between
them.
Insulators
1. Enhancer-Blocking Insulators:
1. Positioned between an enhancer and a promoter.
2. Prevent enhancers from inappropriately activating nearby genes.
3. Function by blocking DNA looping that brings enhancers to non-target
Function Description
promoters.

2. Barrier Insulators:
1. Located at the borders of heterochromatin domains.
2. Recruit chromatin modifiers or nucleosome remodeling complexes that maintain
open chromatin.
3. Prevent heterochromatin spread, protecting active genes from being silenced.

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 4.
Insulators

Schematic diagram summarizing the properties of

insulators and barrier sequences

Alberts (Molecular Biology of the Cell)

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 4.
Insulators

Model of multiple insulator action.

Weaver (Molecular Biology)

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