Regulation of Gene expression

Dr. Shyama Prasad Sajankila, PhD

Associate Professor
Department of Biotechnology Engineering
NMAM Institute of Technology, Nitte, Karkala.

e.mail: [email protected]
[email protected]
Differences between prokaryotes and eukaryotes:

• Prokaryote gene expression typically is regulated by an operon,

the collection of controlling sites adjacent to polycistronic protein
-coding sequences.

• Eukaryotic genes also are regulated in units of protein-coding

sequences and adjacent controlling sites, but operons are not
known to occur.

• Eukaryotic gene regulation is more complex because eukaryotes

possess a nucleus.

(transcription and translation are not coupled).

• Two “categories” of eukaryotic gene regulation exist:

Short-term - genes are quickly turned on or off in response to

the environment and demands of the cell.

Long-term - genes for development and differentiation.

Eukaryote gene expression is regulated at six levels:

1. Transcription

2. RNA processing

3. mRNA transport

4. mRNA translation

5. mRNA degradation

6. Protein degradation / Modification

Transcription control of gene regulation is controlled by:

1. Promoters

• Occur upstream of the transcription start site.

• Some determine where transcription begins (e.g.,

TATA), whereas others determine if transcription
begins.

• Promoters are activated by specialized transcription

factor (TF) proteins (specific TFs bind specific
promoters).

• One or many promoters (each with specific TF

proteins) may occur for any given gene.

• Promoters may be positively or negatively regulated.

1. Transcription control of gene regulation is controlled by:

2. Enhancers
• Occur upstream or downstream of the transcription start
site.

• Regulatory proteins bind specific enhancer sequences;

binding is determined by the DNA sequence.

• Loops may form in DNA bound to TFs and make contact

with upstream enhancer elements.

• Interactions of regulatory proteins determine if

transcription is activated or repressed (positively or
negatively regulated).
Activation of transcription
By transcription factors (TFs),
activator, and coactivator
proteins.
More about promoters and enhancers:
• Some regulatory proteins are common in all cell types,
others are specific.

• Each promoter and enhancer possesses a specific set of

proteins (co-activators) that determines expression.

• Rate of gene expression is controlled by interaction between

positive and negative regulatory proteins.

• Combinatorial gene regulation; enhancers and promoters

bind many of the same regulatory proteins, implying lots of
interaction with fine and coarse levels of control.
Chromatin remodeling:
Acetylation of histones enhances access to promoter
region and facilitates transcription.
DNA methylation and transcription control:
• Small percentages of newly synthesized DNAs (~3% in
mammals) are chemically modified by methylation.

• Methylation occurs most often in symmetrical CG sequences.

• Transcriptionally active genes possess significantly lower levels

of methylated DNA than inactive genes.

• A gene for methylation is essential for development in mice

(turning off a gene also can be important).

• Methylation results in a human disease called fragile X

syndrome; FMR-1 gene is silenced by methylation.
Methylation of H19 inactivates transcription
(involved in expression of insulin like growth factor)
 Hormone regulation - another good example of
short-term regulation of transcription:

• Cells of higher eukaryotes are specialized and

generally shielded from rapid changes in the external
environment.

• Hormone signals are one mechanism for regulating

transcription in response to demands of the
environment.

• Hormones act as inducers produced by one cell and

cause a physiological response in another cell.

• Hormones act only on target cells with hormone

specific receptors, and levels of hormones are
maintained by feedback pathways.
Fig. 18.17, Model of glucocorticoid steroid hormone regulation.
2. RNA processing control:
• RNA processing regulates mRNA production from precursor
RNAs.

• Two independent regulatory mechanisms occur:

• Alternative polyadenylation = where the polyA tail

is added

• Alternative splicing = which exons are spliced

• Alternative polyadenylation and splicing can occur together.

Examples:

• Human calcitonin (CALC) gene in thyroid and neuronal

cells

• Sex determination in Drosophila

Alternative polyadenylation and splicing of the human CACL gene in
thyroid and neuronal cells.

Calcitonin gene
-related
peptide
3. mRNA transport control:
• Eukaryote mRNA transport is regulated.

• Some experiments show ~1/2 of primary

transcripts never leave the nucleus and are
degraded.

• Mature mRNAs exit through the nuclear pores.

4. mRNA translation control:
• Unfertilized eggs are an example, in which mRNAs (stored in
the egg/no new mRNA synthesis) show increased translation
after fertilization).

• Stored mRNAs are protected by proteins that inhibit

translation.
5. mRNA degradation control:
• All RNAs in the cytoplasm are subject to
degradation.

• tRNAs and rRNAs usually are very stable; mRNAs

vary considerably (minutes to months).

• Stability is a major regulatory control point.

• Various sequences and processes affect mRNA half

-life:

• AU-rich elements
• Secondary structure
• Deadenylation enzymes remove As from poly(A) tail
• 5’ de-capping
• Internal cleavage of mRNA and fragment degradation
6. Post-translational control - protein
degradation:
• Proteins can be short-lived (e.g., steroid receptors) or long-
lived (e.g., lens proteins in your eyes).

• Protein degradation in eukaryotes requires a protein co-

factor called ubiquitin. Ubiquitin binds to proteins and
identifies them for degradation by proteolytic enzymes.

• Amino acid at the N-terminus is correlated with protein

stability and determines rate of ubiquitin binding.

• Arg, Lys, Phe, Leu, Trp 1/2 life ≤3

minutes

• Cys, Ala, Ser, Thr, Gly, Val, Pro, Met 1/2 life ≥ 20 hours
Summary and contrasts:

Prokaryotes control expression by:

Transcription

Eukaryotes control expression by:

Transcription

RNA processing

mRNA transport

mRNA translation

mRNA degradation

Protein degradation
4Gene Expression
– Constitutive, Inducible, and Repressible
– Positive and Negative Control

4Operons:
– The Lactose Operon in E. coli: Induction and Catabolite
Repression
– The Tryptophan Operon in E. coli: Repression and
Attenuation

4Translational Control of Gene Expression

4Post-Translational Regulatory Mechanisms

 Regulation of Gene Expression in
Prokaryotes

4Mechanisms that involve the rapid turn-on and turn-

off of gene expression in response to environmental
changes. “Plasticity---------adaptability”

4Preprogrammed circuits or cascades or system or

pathways of gene
Expression. “Proper sequence of events”
 Gene Expression:
Constitutive, Inducible, and
Repressible

Constitutive: Genes that specify cellular components that

perform housekeeping functions— for example, the
ribosomal RNAs and proteins involved in protein synthesis
—are expressed constitutively.

Non-Constitutive: Other genes often are expressed only

when their products are required for growth.
 Constitutive Genes
4 Certain gene products (e.g., tRNAs, rRNAs, ribosomal proteins,
DNA and RNA polymerase subunits, ATP and GTP, enzymes
catalyzing housekeeping functions) are essential components
of almost all living cells.

4 Genes that specify products of this type are continuously

expressed in most cells.
Non-Constitutive Genes: Inducible and
Repressible Genes

4Other gene products are needed for cell

growth only under certain environmental
conditions (aerobics and anaerobics).

4Regulatory mechanisms allow the synthesis

of these gene products only when they are
needed. Why? Energy
Induction of Genes for Lactose
Utilization
4 Gene expression is induced
when glucose is absent and
lactose is present.

4 Induction occurs at the level of

transcription and alters the rate
of enzyme synthesis.

4 Enzymes involved in catabolic

pathways are often inducible.
The kinetics of β-galactosidase induction
Repression of Genes for Tryptophan
Biosynthesis

4 Genes are turned on

(depressed) in the absence of
tryptophan and turned off
(repressed) when tryptophan
is available.

4 Repression occurs at the

level of transcription.

4 Enzymes involved in anabolic

pathways are often
repressible.
 Gene Expression:
The product of a regulatory gene is required
to initiate (turn on) the expression of one or
more genes.

The product of a regulatory gene is required

to turn off the expression of one or more
genes.
 Regulatory gene:
Organization of a bacterial operon
 Positive and Negative Control
Mechanisms

4Regulator genes encode products that regulate the

expression of other genes.

4In positive control mechanisms, the product of the

regulator gene is required to turn on the expression
of structural genes.

4In negative control mechanisms, the product of the

regulator gene is necessary to shut off the
expression of structural genes.
4The product of the regulator gene acts ( regulator
protein) by binding to a site called the regulator
protein binding site (RPBS) adjacent to the promoter
of the structural gene(s).

4In positive control systems, the regulator gene

products are called activators because they activate
transcription of the structural gene(s).

4In negative control systems, the regulator gene

products are called repressors because they
repress transcription of the structural gene(s).
 Effector Molecules
4Whether or not a regulator protein can bind
to the RPBS depends on the presence or
absence of effector molecules in the cell.

4Inducers are effector molecules involved in

induction of gene expression.

4Co-repressors are effector molecules

involved in repression of gene expression.
 Allosteric Transitions
4Effector molecules bind to regulator gene
products (regulator protein) and cause
conformational changes in protein structure
called allosteric transitions.

4Allosteric transitions caused by the binding of

effector molecules to activators and
repressors alter their ability to bind to the
RPBS.
Regulation of Inducible Systems

(Effector)
Regulation of Repressible Systems

(Effector)
 Regulatory Mechanisms
4 In a positive control mechanism, the activator is
involved in turning on gene expression.

4 In a negative control mechanism, the co-repressor

is involved in turning off gene expression.

4 With both positive and negative control mechanisms,

whether gene expression is inducible or repressible
depends on whether the free regulator protein/ or
the regulator protein/ effector molecule complex
binds to the RPBS.
• Gene expression is controlled by both positive and negative
regulatory mechanism.

• In positive control mechanisms, the product of a regulator gene,

an activator, is required to turn on the expression of the
structural gene(s).

• In negative control mechanisms, the product of a regulator

gene, a co-repressor, is required to turn off the expression of
the structural gene(s).

• Activators and repressors regulate gene expression (protein) by

binding to sites adjacent to the promoters of structural genes.

• The effector molecules are called inducers in inducible systems

and co-repressors in repressible systems.
THE lac OPERON
 Operons:
Units required for Gene Expression

Sequence of DNA

In prokaryotes, the operon

includes structural genes, the
operator and the promoter.
 Regulatory gene:
Organization of a bacterial operon
 Control of Gene Expression in Bacteria
4The Bacterial Operon: An operon is a functional
complex of genes containing the information for enzymes of a
metabolic pathway.

4 It includes:
• Structural genes – code for the enzymes and are translated from
a single mRNA (Polycistronic).
• Promoter – where the RNA polymerase binds.
• Operator – site next to the promoter , where the regulatory protein
can bind.
• A repressor (~proteins) which binds to a specific DNA sequence to
determine whether or not a particular gene is transcribed.
• The regulatory gene encodes the repressor protein
The Operon Model

Each operon
contains
--several contiguous
structural genes,

--a promoter

--an operator
The Structural Genes of an Operon
4A single mRNA transcript carries the coding
information of an entire operon.

4Operons containing more than one structural gene

are multigenic.

4All structural genes in an operon are co-transcribed

and therefore are coordinately expressed.
The Lactose (lac) Operon in E. coli:
Induction and Catabolite
Repression

The structural genes in the lac operon

are transcribed only when lactose is
present and glucose is absent.
 The control of gene expression
n Each cell in the human contains all the genetic
material for the growth and development of a human

n Some of these genes will be need to be expressed

all the time

n These are the genes that are involved in of vital

biochemical processes such as respiration

n Other genes are not expressed all the time

n They are switched on an off at need

 Operons
n An operon is a group of
genes that are transcribed at
the same time.

n They usually control an

important biochemical
process.
Jacob, Monod & Lwoff
n They are only found in © NobelPrize.org

prokaryotes.
 The lac Operon
 The lac operon consists of three genes each
involved in processing the sugar lactose

 One of them is the gene for the enzyme β-

galactosidase

 This enzyme hydrolyses lactose into glucose and

galactose
 Adapting to the environment
n E. coli can use either glucose, which is a
monosaccharide, or lactose, which is a disaccharide

n However, lactose needs to be hydrolysed (digested)

first

n So the bacterium prefers to use glucose when it can

 Four situations are possible
1. When glucose is present and lactose is absent the E. coli
does not produce β-galactosidase.

2. When glucose is present and lactose is present the E. coli

does not produce β-galactosidase.

3. When glucose is absent and lactose is absent the E. coli

does not produce β-galactosidase.

4. When glucose is absent and lactose is present the E. coli

does produce β-galactosidase
The control of the lac operon
1. When lactose is absent
n A repressor protein is continuously synthesised. It sits on
a sequence of DNA just in front of the lac operon, the
Operator site
n The repressor protein blocks the Promoter site where
the RNA polymerase settles before it starts transcribing

Repressor RNA
protein Blocked polymerase

DNA
I
O z y a
Regulator Operator
lac operon
gene site
© 2007 Paul Billiet ODWS
 2. When lactose is present
n A small amount of a sugar allolactose is formed within
the bacterial cell. This fits onto the repressor protein at
another active site (allosteric site)
n This causes the repressor protein to change its shape (a
conformational change). It can no longer sit on the
operator site. RNA polymerase can now reach its
promoter site

DNA

I O z y a
© 2007 Paul Billiet ODWS
2. When lactose is present
n A small amount of a sugar allolactose is formed within
the bacterial cell. This fits onto the repressor protein at
another active site (allosteric site)
n This causes the repressor protein to change its shape (a
conformational change). It can no longer sit on the
operator site. RNA polymerase can now reach its
promoter site

DNA
I O z y a
Promotor site
© 2007 Paul Billiet ODWS
3. When both glucose and
lactose are present
n This explains how the lac operon is
transcribed only when lactose is present.
n BUT….. this does not explain why the
operon is not transcribed when both
glucose and lactose are present.

© 2007 Paul Billiet ODWS

 n When glucose and lactose are present RNA
polymerase can sit on the promoter site but it is
unstable and it keeps falling off

Repressor
protein removed

RNA
polymerase
DNA
I O z y a
Promotor site
 4. When glucose is absent and
lactose is present
n Another protein is needed, an activator protein. This
stabilises RNA polymerase.
n The activator protein only works when glucose is absent.
n In this way E. coli only makes enzymes to metabolise
other sugars in the absence of glucose
Activator
protein steadies
the RNA
polymerase
Transcription

DNA
I O z y a
Promotor site
© 2007 Paul Billiet ODWS
Summary
Carbohydrates Activator Repressor RNA lac Operon
protein protein polymerase

+ GLUCOSE Not bound Lifted off Keeps falling No

+ LACTOSE to DNA operator site off promoter transcription
site
+ GLUCOSE Not bound Bound to Blocked by No
- LACTOSE to DNA operator site the repressor transcription

- GLUCOSE Bound to Bound to Blocked by No

- LACTOSE DNA operator site the repressor transcription

- GLUCOSE Bound to Lifted off Sits on the Transcription

+ LACTOSE DNA operator site promoter site

© 2007 Paul Billiet ODWS

An Inducible Operon
A Repressible Operon
The lac Operon of E. coli

Structural genes
Reactions Catalyzed by
-Galactosidase
 Induction of the lac Operon
4In the absence of inducer, the repressor binds to the
lac operator and represses transcription of the
structural genes.

4When the repressor binds to inducer, it is released

from the operator, and transcription of the structural
genes is turned on.

4The inducer, allolactose, is derived from lactose in a

reaction catalyzed by -galactosidase.

4The lac I gene encodes a repressor.

 Partial Diploids
Wild type is monoploid

4Partial diploids can be constructed in E. coli using

fertility factors that carry chromosomal genes (F’
factors).

4F’ factors have been used to study the interactions

between different components of the operon.

4Mutations in the structural genes, the operator, and

the repressor can be studied in this way.

4Mutations in the I gene (I–) and the operator (Oc) result

in constitutive synthesis.
 Mutations in the Structural Genes

4The wild-type alleles (I+P+O+Z+Y+A+) of the structural

genes are dominant to the mutant alleles (I-P-O-Z-Y-A-)

4Genotypes F’ I+P+O+Z+Y+A+ / I+P+O+Z-Y-A- and F’

I+P+O+Z-Y-A-/ I+P+O+Z+Y+A+ are inducible for utilization
of lactose.
 lacI+ is Dominant over lacI–

How is this dominance regulated?

cis Dominance of lacI+
 trans Dominance of lacI+

The lacI+ allele encodes a diffusible

product and is dominant in cis and in trans.
 The operator

4The Oc mutant is the binding site for the repressor and

does not encode a product; this mutant acts only in
cis.

4Genotype F’ I+P+OcZ-Y-A- / I+P+O+Z+Y+A+ is inducible

for the structural genes, but genotype F’ I+P+OcZ+Y+A+
/ I+P+O+Z-Y-A- synthesizes the enzymes constitutively.
F’ I+P+OcZ-Y-A- / I+P+O+Z+Y+A+
in the absence of inducer
F’ I+P+OcZ-Y-A- / I+P+O+Z+Y+A+
in the presence of inducer
Constitutive Synthesis in Genotype
F’ I+P+OcZ+Y+A+ / I+P+O+Z-Y-A-
Other Mutations in the lac Operon

4The I-d mutations are dominant to I+.

– The lac repressor acts as a tetramer.
– Heteromultimers containing wild-type and mutant
polypeptides are unable to bind to the operator.

4Is (superrepressed) mutations cause the lac

operon to be uninducible due to low affinity of
the mutant repressor for the inducer.
Catabolite Repression (Glucose effect)
High glucose… low induction of lac operator

4The lac promoter has two components

– The RNA polymerase binding site
– A binding site for catabolite activator protein (CAP)

4Binding of CAP to the promoter activates transcription

of the lac operon from being induced when glucose is
absent.

4CAP binds to the promoter only when cyclic AMP

(cAMP) is present at sufficient concentrations.
Organization of the lac Operon
Promoter-Operator Region
 CAP Exerts Positive Control of the
lac Operon; cAMP is the Effector
4When glucose is 4When glucose is
present absent
– Adenylcyclase is – Adenylcyclase is
inactive. active.
– cAMP levels are low. – cAMP levels are
– CAP cannot bind to high.
the lac operon. – CAP/cAMP binds to
– The lac structural the lac operon.
genes cannot be – The lac structural
induced at high genes can be
levels. induced.
Glucose/ cAMP
 Binding of CAP/cAMP to DNA

Bending and wrapping of DNA by Cap/cAMP: specific sequence is

required for this protein-DNA interaction.
Binding of the lac Repressor to DNA

--Specific binding site for the

repressor (tetramer)

--they bind O1 or (O2 or O3)

forming a DA loop
• The E. coli lac operon is a negative inducible and catabolite
repressible system; the three structural genes in the lac operon
are transcribed at high levels only in the presence of lactose
and the absence of glucose.

• In the absence of lactose, the lac repressor binds to the lac

operator and prevents RNA polymerase from initiating
transcription of the operon.

• Catabolite repression keeps operons such as lac encoding

enzymes involved in carbohydrate catabolism from being
affected (induced) in the presence of glucose, the preferred
energy source.
• The binding of the CAP/cAMP complex to its
binding site in the lac promoter bends the DNA
and makes it more accessible to RNA
polymerase.

• The lac repressor binds to two operators-either

O1 and O2 or O1 and O3-simultaneously and
bends the DNA into a hairpin or a loop,
respectively.
 The Tryptophan Operon in E. coli:
Repression and Attenuation
It controls the synthesis of the enzymes required for the synthesis
of trytophan
The structural genes in the tryptophan operon are transcribed
only when tryptophan is absent or present in low
concentrations.
How is it regulated?
The expression of the genes in the trp operon is regulated by
repression of transcriptional initiation and by attenuation
(premature termination) of transcription when tryptophan is
prevalent in the environment.
Organization of the trp Operon
TrpR: Repressor gene

O: operator region
P1 and P2: promoter
regions

Transcription from TrpL to TrpA

Transcription from TrpC to TrpA

t and t’: Transcription termination

sequence
Absence of tryptophan (W): -derepressed state-
RNA pol binds to P regions and transcribes

Presence of tryptophan (W): -repressed state-

Repressor binds to P regions and preventing
RNA pol binding

TrpR mutants: ? ..still ….. repressed…

New independent mechanism of regulation

(nucleotide sequences located in trpL and the amount of Trp)

 The Leader Peptide and
Attenuator Sequence

Attenuation
-occurs by controlling the termination of transcription close the
mRNA of the leader sequence. It contains G:C rich regions
followed by several A:T bps.

-needs trp (or trp charged-tRNAw).

-produces small transcritp.

Alternate Secondary Structures
of the trpL Transcript
 The Transcription-Termination
Sequence t Forms a Transcription-
Termination Hairpin
 When Tryptophan Levels are Low,
Transcription Proceeds through the Entire
trp Operon
When Tryptophan Levels are high
enough, Transcription Stops at the
Attenuator Sequence
• The E. coli trp operon is a negative repressible
system; transcription of the five structural genes in the
trp operon is repressed in the presence of significant
concentrations of tryptophan.

• Operons such as trp that encode enzymes involved in

amino acid biosynthetic pathways often are controlled
by a second regulatory mechanism called
attenuation.

• Attenuation occurs by the premature termination of

transcription at a site in the mRNA leader sequence
(the sequence 5’ to the coding region) when
tryptophan is prevalent in the environment in which the
bacteria are growing.
Translational Control of Gene
Expression

The regulation of gene expression is often

fine-tuned by modulating either the
frequency of initiation of translation or the rate
of polypeptide chain elongation.

mRNA are multigenic (several genes)

E. Coli Lac operon mRNA (3 enzymes)

Post-Translational Regulatory
Mechanisms
Feedback ( control) inhibition occurs when the
product of a biosynthetic pathway inhibits the
activity of the first enzyme in the pathway,
rapidly shutting off the synthesis of the product.

Rate Limiting
Feedback Inhibition(End-product inhibition)
Trytophan inhibition

allosteric site