Regulation of Gene Expression

Gene Expression
• The central dogma in genetics describes the flow of genetic
information in cells from DNA to mRNA to protein

• Gene expression: is the process by which information from

a gene is used in the synthesis of a functional gene
products: either protein or RNA such as tRNA and rRNA
Gene Expression
• Gene regulation control cell structure and function. It is the basis
for cellular division, differentiation and morphogenesis

Cell Proliferation Cell Specialization Cell movement and morphogenesis

• Different cell types differ dramatically in both structure and

function although they contain the same genome (e.g. basophil
and neuronal cell)

basophil Neuronal cell

Gene Expression
• Different cell types synthesize and accumulate different sets
of RNA and proteins (Hemoglobin in RBCs)
• Also, the level of expression of almost every active gene
varies from one cell type to another

• Gene expression can be regulated at many steps:

1. Transcriptional control (the most efficient point of gene
expression regulation)
2. RNA processing control
3. RNA transport and localization control
4. Translational control
5. mRNA degradation control
6. Protein activity control
Steps in Eukaryotic Gene Expression
regulation
1. Transcription Initiation
• Steps: Initiation, elongation and termination
• RNA polymerase catalyzes the synthesis of RNA strand
from DNA template.
• The promoter is a regulatory DNA region (100-1000 bp). In
eukaryotes, it consists of consensus sequences such as
TATA box, BRE, INR and DPE . In prokaryotes, two
consensus sequences at - 10 and -35.

Start point
-35 -10 +1
prokaryotic promoter
TATA
box
1. Transcription Initiation
Transcription initiation in
eukaryotes Transcription initiation in
prokaryotes
1. It requires general transcription
factors which assemble together 1. σ factor recognizes the -35
with RNA polymerase at the region in the promoter and
promoter to form pre-initiation binds to it
complex (PIC) 2. Once the RNA polymerase
2. TFIID binds first at the TATA box starts the transcription, σ factor
via its TBP subunit then dissociates to guide
another enzyme to the initiation
site.
 Regulation of Transcription Initiation

• Gene regulatory proteins called specific

transcription factors (activators or repressors)
bind DNA specific sequences called gene
regulatory regions (enhancers or silencers) to
control the expression of various genes
• Specific transcription factors (regulatory
proteins) are different from general transcription
factors which are involved in the transcription
initiation process
Regulation of Transcription Initiation

General transcription factors

RNA polymerase II
Regulation of Transcription Initiation

• Cis-regulatory elements (CREs): are regions of

non-coding DNA which regulate the transcription of
nearby genes. These include promoters,
enhancers and silencers
• Gene regulatory proteins act at distance: DNA
looping allows them to interact with the assembled
proteins at the promoter
• Mediator is a protein complex recruited to the
promoter via specific transcription factors. It
provides extended contact area for the gene
regulatory proteins
Regulation of Transcription Initiation
Regulation of Transcription Initiation

recruited by repressor

recruited by activator
Regulation of Transcription Initiation
Regulation of Transcription Initiation

General transcription factors

RNA polymerase II
 Regulation of Transcription Initiation

• Beside the mediator, other proteins are recruited by

specific transcription factors to the promoter such
as: histone modifying enzymes and chromatin
remodeling complexes
• Epigenetic factors: gene expression is affected by
changes in chromatin structure (Heterochromatin/
Euchromatin)
Regulation of Transcription Initiation
in Prokaryotes
• The expression of many genes is regulated according to the
available food in the environment
• Operon: DNA unit consists of a cluster of related genes
controlled by single promoter and transcribed together into
single mRNA strand (bicistronic or polycistronic transcript)
• Operator: a segment of regulatory DNA to which a
repressor can bind to regulate the transcription of
downstream target genes
• The three basic DNA components of operon:
1. Promoter
2. Operator
3. Structural genes
• Examples in E-coli bacteria: Trp operon and Lac operon
 Trp Operon
• Trp operon consists of five structural genes required
for the biosynthesis of the amino acid tryptophan

Polycistronic
transcript
Trp Operon

(Co-repressor)
Trp Operon
• Trp operon consists of five structural genes required for the
biosynthesis of the amino acid tryptophan

• In the presence of tryptophan in the growth medium, trp

repressor (a gene regulatory protein) binds the operator
and blocks the access of RNA polymerase (negative
control)
• In the absence of tryptophan, the repressor is in the
inactive form so cannot bind the operator and the enzymes
are transcribed as single polycistronic mRNA
Lac Operon
• Lac operon consists of three structural genes required for
the transport and metabolism of lactose as an alternative
carbon source to glucose:

1. lacZ : encodes β-galactosidase which cleaves lactose

into glucose and galactose
2. lacY : encodes lactose permease to transport lactose
into the cell
3. lacA : encodes galactoside O-acetyltransferase which
plays a role in cell detoxification
Lac Operon
binding site
binding site

Allolactose (Co-repressor)

(gene regulatory protein) (Co-activator)

(gene regulatory protein)

Lac Operon
• It is under both negative and positive transcriptional
controls (dual control): the lac repressor and the
CAP activator (catabolite activator protein)
respectively
Lac Operon

binding site
Lac Operon
Lac Operon

binding site

Allolactose
Lac Operon

Allolactose
Lac Operon
• It is under both negative and positive transcriptional
controls (dual control): the lac repressor and the CAP
activator (catabolite activator protein) respectively
• In the absence of lactose, lac repressor binds lac operator
and inhibits RNA polymerase binding so genes are
switched off (regardless of glucose level).
• In the absence of glucose, cAMP level is high. cAMP is co-
activator of CAP.
• In presence of lactose, the lac repressor is inactivated by
the binding to lactose isomer “allolactose” so the repressor
dissociates from the operator with the genes are weakly
transcribed in the presence of glucose but extensively
transcribed in the absence of glucose
• Allolactose is an inducer of lac operon. It acts as co-
repressor of lac repressor protein
2. RNA Splicing Control
• The post transcriptional modification (5’-capping, splicing
and 3’ polyadenylation) transfers pre-mRNA into the mature
transcript mRNA (Eukaryotes only)
(5’ splice site) (3’ splice site)

• RNA splicing is the

removal of introns
and the joining of
exons catalyzed by
spliceosome
(RNA-protein complex)
2. RNA Splicing Control
• Alternative RNA splicing (splicing at different junctions)
produces different forms (isoforms) of a protein from the
same gene
2. RNA Splicing Control
• Many genes undergo alternative splicing in tissue-specific
manner and/or under specific cellular conditions (a protein
is nonfunctional in one cell type but functional in another
cell type)
• Alternative splicing of pre-mRNA transcripts is regulated by
proteins (activators/repressors) that bind specific RNA
sequences (regulatory elements) on the primary transcript
itself
3. Editing of RNA
• RNA editing creates mature mRNAs that are not truly
encoded by the genome (it occurs in the pre-mRNA)
• Two types of editing in mammals:
1. Deamination of A to I (inosine)
2. Deamination of C to U

• The editing occurring in coding region produces

protein isoforms but that occurring outside coding
region affects the pattern of pre-mRNA splicing,
transport of mRNA from nucleus to cytosol or
efficiency of RNA translation
3. Editing of RNA
• RNA editing is tissue-specific. For example, Apolipoprotein
B exists in 2 isoforms: ApoB48 expressed in small intestine
(produced by C to U editing to create
premature stop codon UAA
instead of glutamine
codon CAA) and
ApoB100 expressed
in the liver

• ApoB48 lacks the

C-terminal part of
ApoB100
4. RNA Transport & Localization Control
• Gene expression can be regulated by controlling the
nuclear transport of mRNAs and their localization to specific
cytoplasmic domains
• Processing of pre-mRNA in
the nucleus is critical for the
cytoplasmic localization
• The cellular address is
specified by cis-acting
elements (called localization
elements) mostly found in
3’UTR
• Localization elements are
recognized by trans-acting
factors (RNA-binding proteins)
5. Translation Control
• Initiation, elongation and termination
• Regulation of translation initiation is one determinant
of the rate at which any protein is synthesized
• The mechanism of translation initiation is different
between eukaryotes and prokaryotes
Translation Initiation in Eukaryotes
1. Assembly of 43S pre-initiation complex (PIC) facilitated by eukaryotic
initiation factors (eIFs)
2. Activation of 5’ end of mRNA by eIF4E and eIF4G
3. Binding of PIC to start scanning for initiating AUG codon
4. Dissociation of eIFs and binding of large ribosomal subunit
Translation Initiation in Prokaryotes
1. Binding of the small ribosomal subunit to the Shine Dalgarno sequence

2. Recruitment of initiator tRNA

carrying formylmethionine

3. Dissociation of IFs and

binding of large ribosomal subunit
5. Translation Control

• Initiation, elongation and termination

• Regulation of translation initiation is one determinant of the
rate at which any protein is synthesized
• The mechanism of translation initiation is different between
eukaryotes and prokaryotes
• There are several mechanisms of translation initiation
regulation:
1. Repressor proteins: bind to
and block the Shine-Dalgarno
sequence. Similarly in eukaryotes,
translational repressors bind 5’ end
of mRNA or 3’ UTR
5. Translation Control
2. Antisense RNA: a short RNA sequence (miRNAs)
base pairs with specific region of
mRNA (near the AUG start codon)
and blocks its translation. Morpholino
is a widely used technique for
gene silencing

3. Phosphorylation of eIF2 by
kinases. eIF2
P traps the
guanine nucleotide
exchange factor eIF2B
and prevents the reuse
of nonphosphorylated
eIF2 and greatly slows
protein synthesis
6. mRNA Degradation Control

• Changes in mRNA stability can regulate gene

expression
• Eukaryotic mRNAs are more stable (half-life 30min-
10hrs) than bacterial mRNAs (half life < couple of
minutes)
• 3’ UTR sequences carry binding sites for proteins
that increase or decrease the degradation rate.
6. mRNA Degradation Control

• For example, 3’ UTR of transferrin

receptor mRNA contains a
recognition site for
endonucleases. This site
can be blocked or kept exposed in
response to extracellular signals
likethe availability of iron which
controls the expression of
transferrin receptor via regulating
the binding or dissociation of
aconitase (an iron-sensitive RNA
binding protein)
7. Post Translational Control
• For a protein to be functional, it should be correctly folded
• Mis-folded proteins are tagged with ubiquitin molecules for
degradation with proteasome mechanism (protease
enzymes)
• Accumulation of aberrant proteins leads to cell damage
The Fate of Proteins after translation
Ubiquitination/ Ubiquitylation
• Ubiquitin tag is added to the target
protein in an ATP-dependent
reaction to produce poly-
ubiquitinated protein

• The first ubiquitin residue is

covalently added by ligase
enzyme. This enhances the
addition of several tags

• The degradation process can

be regulated. It can be induced
by activation of ubiquitin ligase or
activation of degradation signal found
at N-terminus of target
protein