Regulation of Gene Expression-1

(In prokaryotes)

By- Professor (Dr.) Namrata Chhabra

Biochemistry For Medics- Lecture Notes
www.namrata.co
 Introduction
o Gene expression is the combined process of
the transcription of a gene into mRNA,
o the processing of that mRNA, and
o its translation into protein (for protein-
encoding genes).

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 Significance of gene Expression

Regulated expression of genes is required for

Adaptation,
Differentiation and
Development

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 1) Adaptation
1) Adaptation- Organisms adapt to
environmental changes by altering gene
expression.
a) Bacteria are highly versatile and responsive
organisms: the rate of synthesis of some
proteins in bacteria may vary more than a
1000-fold in response to the supply of
nutrients or to environmental challenges.

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 1) Adaptation

b) Cells of multicellular organisms also respond

to varying conditions.
Such cells exposed to hormones and growth
factors change substantially in shape, growth
rate, and other characteristics.

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 2) Tissue specific differentiation and
development
The genetic information present in each
somatic cell of a metazoan organism is
practically identical.
The exceptions in the genetic information are
found in those few cells that have amplified or
rearranged genes in order to perform
specialized cellular functions.

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 2) Tissue specific differentiation and
development
Cells from muscle and nerve tissue show
strikingly different morphologies and other
properties, yet they contain exactly the same
DNA.
These diverse properties are the result of
differences in gene expression.
Expression of the genetic information is
regulated during ontogeny and differentiation
of the organism and its cellular components.

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 Control of gene Expression
• Mammalian cells possess about 1000 times more
genetic information than does the bacterium
Escherichia coli.
• Much of this additional genetic information is
probably involved in regulation of gene
expression during the differentiation of tissues
• and biologic processes in the multicellular
organism and in ensuring that the organism can
respond to complex environmental challenges.
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 How is gene expression controlled?

• Gene activity is controlled first and foremost

at the level of transcription.
• Much of this control is achieved through the
interplay between proteins that bind to
specific DNA sequences and their DNA binding
sites.
• This can have a positive or negative effect on
transcription.

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 How is gene expression controlled?

• Transcription control can result in tissue-

specific gene expression.
• In addition to transcription level controls,
gene expression can also be modulated by
gene amplification, gene rearrangement,
posttranscriptional modifications, and RNA
stabilization.

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 Types of gene regulation

There are three types of genes regulation-

• Positive
• Negative and
• Double negative

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 Types of gene regulation
A) Positive regulation
• When the expression of genetic information is
quantitatively increased by the presence of a
specific regulatory element, regulation is said
to be positive.
• The element or molecule mediating positive
regulation is a positive regulator or activator.

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 Types of gene regulation
B) Negative regulation
• When the expression of genetic information is
diminished by the presence of a specific
regulatory element, regulation is said to be
negative.
• The element or molecule mediating negative
regulation is said to be a negative regulator or
repressor.

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 Types of gene regulation
A double negative has the effect of acting as a
positive.
• An effector that inhibits the function of a
negative regulator will bring about a positive
regulation.
• Many regulated systems that appear to be
induced are in fact derepressed at the
molecular level.

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 Responses in Gene Expression
• Type A response is characterized by an
increased extent of gene expression that is
dependent upon the continued presence of
the inducing signal.
• When the inducing signal is removed, the
amount of gene expression diminishes to its
basal level,
• The amount repeatedly increases in response
to the reappearance of the specific signal.

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 Type A response

The response is observed only in the presence

of a signal
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 Type A response

• This type of response is commonly observed

in prokaryotes in response to sudden changes
of the intracellular concentration of a
nutrient.
• It is also observed in many higher organisms
after exposure to inducers such as hormones,
nutrients, or growth factors

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 Type B Response

• Type B response exhibits an increased amount of

gene expression that is transient even in the
continued presence of the regulatory signal.
• After the regulatory signal has terminated and
the cell has been allowed to recover, a second
transient response to a subsequent regulatory
signal may be observed.

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 Type B Response

The signal persists but the response is

transient.
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 Type B Response
• This phenomenon of response-
desensitization-recovery characterizes the
action of many pharmacologic agents, but it is
also a feature of many naturally occurring
processes.
• This type of response commonly occurs
during development of an organism, when
only the transient appearance of a specific
gene product is required although the signal
persists.
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 Type C Response
• The type C response pattern exhibits, in
response to the regulatory signal,
• an increased extent of gene expression that
persists indefinitely even after termination of
the signal.
• The signal acts as a trigger in this pattern.

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 Type C Response

• The response is signal independent.

• Response persists even in the absence of a
signal.
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 Type C Response
• Once expression of the gene is initiated in the
cell, it cannot be terminated even in the
daughter cells;
• It is therefore an irreversible and inherited
alteration.
• This type of response typically occurs during
the development of differentiated function in
a tissue or organ.

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Types of genes in Gene Expression
• Inducible gene- An inducible gene is one
whose expression increases in response to an
inducer or activator, a specific positive
regulatory signal.
• Inducible genes have relatively low basal rates
of transcription.

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Types of genes in Gene Expression
• Constitutive genes-are expressed at a
reasonably constant rate and are not known
to be subjecedt to regulation.
• These are often referred to as housekeeping
genes.

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Types of genes in Gene Expression

• As a result of mutation, some inducible gene

products become constitutively expressed.
• A mutation resulting in constitutive expression
of what was formerly a regulated gene is
called a constitutive mutation.

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 Regulation of Prokaryotic Gene
Expression
• Controlling gene expression is one method of
regulating metabolism.
• Prokaryotes must use substances and
synthesize macromolecules just fast enough
to meet their needs.

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 Regulation of Prokaryotic Gene
Expression
• The genes for metabolizing enzymes are
expressed only in the presence of nutrients.
• If the enzymes are not needed, genes are
turned off.
• This allows for conservation of cell resources.

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Features of Prokaryotic gene
Expression
• In prokaryotes, the genes involved in a
metabolic pathway are often present in a
linear array called an Operon, e.g., the lac
Operon.
• An Operon can be regulated by a single
promoter or regulatory region.
• The cistron is the smallest unit of genetic
expression.
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 Cistron
• Some enzymes and other protein molecules
are composed of two or more non identical
subunits.
• The "one gene, one enzyme" concept is not
necessarily valid.
• The cistron is the genetic unit coding for the
structure of the subunit of a protein molecule

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 Cistron
• A single mRNA carries information for multiple
proteins
• This type of mRNA is called a polycistronic
mRNA and is totally unique to prokaryotes
• The polycistronic Lac Operon mRNA is
translated into three separate proteins
• In Eukaryotes the m-RNA is monocistronic

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 Lac Operon Model
• Jacob and Monod in 1961 described their
Operon model in a classic paper.
• Their hypothesis was to a large extent based
on observations on the regulation of lactose
metabolism by the intestinal bacterium E coli.

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 Lac Operon – Basic concept
• Bacteria such as E. coli usually rely on glucose
as their source of carbon and energy.
• However, when glucose is scarce, E. coli can
use lactose as their carbon source even
though this disaccharide does not lie on any
major metabolic pathways.
• An essential enzyme in the metabolism of
lactose is β-galactosidase, which hydrolyzes
lactose into galactose and glucose
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 Lac Operon – Basic concept

Action of Beta galactosidase on lactose,

breaks lactose to galactose and glucose

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 Inducible Lac Operon
• An E. coli cell growing on a carbon source such
as glucose or glycerol contains fewer than 10
molecules of β -galactosidase.
• In contrast, the same cell contains several
thousand molecules of the enzyme when
grown on lactose.
• The presence of lactose in the culture medium
induces a large increase in the amount of β
-galactosidase by eliciting the synthesis of new
enzyme molecules rather than by activating a
preexisting but inactive precursor.
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 Components of Lac Operon
• The genetic elements of the model are a
regulator gene, a regulatory DNA sequence
called an operator site, and a set of structural
genes.
• The regulator gene encodes a repressor protein
that binds to the operator site.
• The binding of the repressor to the operator
prevents transcription of the structural genes.
• The operator and its associated structural
genes constitute the Operon.
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 Components of Lac Operon
• For the lactose (lac) Operon, the i gene
encodes the repressor, o is the operator site,
and the z, y, and a genes are the structural
genes for β -galactosidase, the permease, and
the transacetylase, respectively.
• The Operon also contains a promoter site
(denoted by p), which directs the RNA
polymerase to the correct transcription
initiation site. transcript.
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 Components of Lac Operon

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 Components of Lac Operon

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 Components of Lac Operon

•The z, y, and a genes are transcribed to give a

single mRNA molecule that encodes all three
proteins.
• An mRNA molecule encoding more than one
protein is known as a polygenic or polycistronic

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 How does the lac repressor inhibit
the expression of the lac Operon?
•The lac repressor can exist as a dimer of 37-kd subunits,
and two dimers often come together to form a
tetramer.
• In the absence of lactose, the repressor binds very
tightly and rapidly to the operator.

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 Negative Control- Repression

•When the lac repressor is bound to DNA, it

prevents bound RNA polymerase from locally
unwinding the DNA to expose the bases that
will act as the template for the synthesis of the
RNA strand.
•Thus, very little β-galactosidase, permease, or
transacetylase are produced.

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 Promoter Operator
Lac I Lac Z Lac Y Lac A
gene gene
RNA polymerase

Translation &
Transcription No Gene
Expression

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 How does the presence of lactose trigger
expression from the lac Operon?
(b) Double negative control- Derepression
Lactose or lactose analogue, bind to lac
repressor and act as inducers of lac Operon
A lactose analog that is capable of inducing
the lac Operon while not itself serving as a
substrate for -galactosidase is an example of
a gratuitous inducer. An example is
isopropylthiogalactoside (IPTG)

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(b) Double negative control- Derepression
•When the lac repressor is bound to the inducer,
the repressor's affinity for operator DNA is greatly
reduced.
•This binding leads to local conformational
changes so that it cannot easily contact DNA
simultaneously, leading to a dramatic reduction in
DNA-binding affinity and the release of DNA by
the lac repressor.
•With the operator site unoccupied, RNA
polymerase can then transcribe the other lac
genes and the bacterium produces the proteins
necessary for the efficient utilization of lactose.
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 Promoter Operator
Lac I Lac Z Lac Y Lac A
gene gene
RNA polymerase

Translation &
Transcription
Desired
product

R
Repressor Inactive
tetramer
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 (b) Double negative control- Derepression

An inducer derepresses the lac Operon and

allows transcription of the structural genes for β-
galactosidase, galactoside permease, and
thiogalactoside transacetylase.
 Repressible and Inducible enzymes are both an
example of negative control of a pathway.
 Activating the repressor proteins shuts off the
pathway.
 Positive control requires that an activator
molecule switch on transcription.
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 (

c) Positive control- CAP-cAMP

binding
There are also DNA-binding proteins that
stimulate transcription.
One particularly example is the catabolite
activator protein (CAP), which is also known as the
cAMP response protein (CRP).
Within the lac Operon, CAP binds to an inverted
repeat that is centered near position -61 relative to
the start site for transcription

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c) Positive control- CAP-cAMP binding

Within the lac Operon, CAP binds to an inverted

repeat that is centered near position -61 relative
to the start site for transcription
The CAP-cAMP complex stimulates the initiation
of transcription by approximately a factor of 50.
A major factor in this stimulation is the
recruitment of RNA polymerase to promoters to
which CAP is bound.

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c) Positive control- CAP-cAMP binding

An increase in the cAMP level

inside an E. coli bacterium results
in the formation of CAP-cAMP
complexes that bind to many
promoters and stimulate the
transcription of genes encoding a
variety of catabolic enzymes.
Thus, the CAP-cAMP regulation
acts as a positive regulator
because its presence is required
for gene expression.
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State of Lac Operon in the presence
of only glucose
When grown on glucose, E. coli have a very
low level of catabolic enzymes such as β-
galactosidase.
 It would be wasteful to synthesize these
enzymes when glucose is abundant.
The inhibitory effect of glucose, called
catabolite repression, is due to the ability of
glucose to lower the intracellular
concentration of cyclic AMP.
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State of Lac Operon in the presence of
only glucose
The bacterium accumulates cAMP only when it is
starved for a source of carbon.
 In the presence of glucose—or of glycerol in
concentrations sufficient for growth—the bacteria
will lack sufficient cAMP to bind to CAP because the
glucose inhibits adenylyl cyclase, the enzyme that
converts ATP to cAMP.
Thus, in the presence of glucose or glycerol,
cAMP-saturated CAP is lacking, so that the DNA-
dependent RNA polymerase cannot initiate
transcription of the lac Operon.
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Maximum Expression of Lac Operon
The lac Operon is controlled by two
distinct DNA binding factors;
 One that acts positively (cAMP-CRP
complex) and
The other that acts negatively (LacI
repressor).
Maximal activity of the lac Operon occurs
when glucose levels are low (high cAMP
with CAP activation) and lactose is presen,t
LacI is prevented from binding to the
operator).
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Constitutive Expression and
continuous repression
When the lacI gene has been mutated so that its product,
LacI, is not capable of binding to operator DNA, the organism
will exhibit constitutive expression of the lac Operon.
 In a contrary manner, an organism with a lacI gene
mutation that produces a LacI protein which prevents the
binding of an inducer to the repressor will remain repressed
even in the presence of the inducer molecule, because the
inducer cannot bind to the repressor on the operator locus in
order to derepress the Operon.
Similarly, bacteria harboring mutations in their lac operator
locus such that the operator sequence will not bind a normal
repressor molecule constitutively express the lac Operon
genes.
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 Promoter Operator
Lac I Lac Z Lac Y Lac A
gene gene
RNA polymerase

mRNA
No Gene
Expression

R R

R R

Repressor Repressor Lactose absent

molecules
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 Promoter Operator
Lac I Lac Z Lac Y Lac A
gene gene
RNA polymerase

Lactose/
Isopropyl
mRNA Thiogalactoside
β-
galactosidase Inducer (IPTG) present
mRNA
R R
R
R R
Permeas Thiogalacto
e side
transacetyl
Inactive repressor
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 If there occurs no glucose metabolism

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 If there occurs glucose metabolism

cAMP

Promoter Operator cAMP ↑↑↑

Lac I Lac Z Lac Y Lac A
gene gene

RNA polymerase
Glucose pool gets depleted
due to metabolism

I
β -galactosidase
CAP-cAMP
R complex formed
Permease mRNA
IR
Thiogalactoside
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 Summary- Regulation of Expression
of Lac Operon

1) In the absence of lactose- Lac Operon remains

repressed due to the presence of lac repressor at
the operator site- (Negative control).
2) In the presence of only Lactose- Lac Operon is
derepressed, the structural genes are transcribed
and the lactose metabolizing enzymes are
synthesized (Double negative control).

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 Summary- Regulation of Expression
of Lac Operon
3) In the presence of both glucose and
lactose- CAP -cAMP complex is not formed,
RNA polymerase can not initiate the
transcription of structural genes despite the
fact that the operator site is vacant due to
the binding of lactose/allolactose with lac
repressor.
Lac Operon remains in the repressed state. It
is absence of positive regulation.
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 Revision of Concepts
1)
http://bcs.whfreeman.com/thelifewire/content/chp13/1302
001.html
2)
http://highered.mcgrawhill.com/sites/0072556781/student_
view0/chapter12/animation_quiz_4.html
3) http://www.youtube.com/watch?
v=iPQZXMKZEfw&feature=related

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