Gene Expression in
Prokaryotes
Why regulate gene expression?

It takes a lot of energy to make RNA and
protein.
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Therefore some genes active all the time
because their products are in constant demand.
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Others are turned off most of the time and are
only switched on when their products are
needed.
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The control of gene expression is much more
complex in eukaryotes than in prokaryotes.
Reasons being, Eukaryotes have:
– Compartmentalization of cells
– More extensive transcript processing
– Regulation from a distance
– Cell and tissue specific gene expression
– Larger Genome size
– Genes scattered about the genome
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In prokaryotes, the control of transcriptional initiation is the
major point of regulation
In eukaryotes the regulation of gene expression is controlled
nearly equivalently from many different points:
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Initiation of transcription (most important control)
Chromatin control
Epigenetic control
Transcript processing
Transcript stability
RNA transport
Protein stability
Protein transport
Post-Translational modifications
Gene Control in Prokaryotes
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One way in which prokaryotes control gene
expression is to group functionally related
genes together so that they can be
regulated together.
This grouping is called an operon.
The clustered genes are transcribed
together from one promoter giving a
polycistronic messenger.
Gene Control in Prokaryotes
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An operon can be defined as a cluster gene that
encode the proteins necessary to perform
coordinated function
Genes of the same operon have related functions
within the cell and are turned on (expressed)
and off together (suppressed).
The first operon discovered was the lac operon
so named because its products are involved in
lactose breakdown.
An operon consists of:
– a promoter (binding site for RNA
polymerase)
– a repressor binding site called an
operator that overlaps the promoter.
– structural genes
Operator
 Repressor proteins encoded by repressor genes, are
synthesized to regulate gene expression.

They bind to the operator site to block transcription
by RNA polymerase.
Promoter
 The promoter sequences are recognized by RNA
polymerase.

When RNA polymerase binds to the promoter,
transcription occurs
Actvators
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The activity of RNA polymerase is also
regulated by interaction with accessory
proteins called activators
The presence of the activator removes
repression and transcription occurs

Two major modes of transcriptional regulation function in
bacteria (E. coli) to control the expression of operons:
– repression and
– induction.
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Both mechanisms involve repressor proteins.
Induction happens in operons that produce gene
products needed for the utilization of energy.
Repression regulates operons that produce gene
products necessary for the synthesis of small biomolecules
such as amino acids.
Inducible system
Negative control
the effector molecule interacts with the repressor protein
such that it cannot bind to the operator

With inducible systems, the binding of the effector
molecule to the repressor:
– greatly reduces the affinity of the repressor for the
operator
– the repressor is released and transcription proceeds.
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In addition to negative control mediated by a
repressor, expression from an inducible
operon is also under positive control,
mediated by an activator
A classic example of an inducible (catabolitemediated) operon is the lac operon, responsible
for obtaining energy from galactosides such as
lactose.
Repressible system
Negative control
the effector molecule interacts with the repressor protein
such that it can bind to the operator

With repressible systems, the binding of the effector
molecule to the repressor:
– greatly increases the affinity of repressor for the
operator
– the repressor binds and stops transcription.

For the trp operon , the addition of tryptophan (the
effector molecule) to the E. coli environment shuts off
the system because the repressors binds at the operator.
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In addition to negative control mediated by a
repressor, expression from a repressible operons is
attenuated by sequences within the transcribed
RNA.
A classic example of a repressible (and attenuated)
operon is the trp operon, responsible for the
biosynthesis of tryptophan.
Structure of the lac Operon

The lac operon three structural genes:
– Z
– y
– a

The z gene codes for β-galactosidase , responsible for the
hydrolysis of the disaccharide, lactose into its monomeric
units, galactose and glucose.
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The y gene codes for permease, which increases
permeability of the cell to galactosides.
The a gene encodes a transacetylase.
In addition to the structural genes the lac operon also has
regulatory genes:
– Promoter: Binding site for RNA polymerase
– Operator: Binding site of repressor
Control of lac operon
expression
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The control of the lac operon occurs by both
positive and negative control mechanisms.
Negative control of the lac operon
What happens to lac operon when glucose is
present and lactose is absent?
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During normal growth on a glucose-based medium
(lacking lactose), the lac repressor is bound to the
operator region of the lac operon, preventing
transcription.
What happens when glucose is absent
and lactose is present?
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The few molecules of lac operon enzymes
present will produce a few molecules of
allolactose from lactose.
Allolactose is the inducer of the lac operon.
The inducer binds to the repressor causing
a conformational shift that causes the
repressor to release the operator.

With the repressor removed, the RNA
polymerase can now bind the
promoter and transcribe the operon.
Positive Control of the lac operon
What happens when both glucose and lactose
levels are high?
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Since the inducer is present, the lac operon will be
transcribed.
However the rate of transcription is very slow (almost
repressed) because glucose levels are high and
therefore cAMP levels are low.
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The repression of the lac operon under these conditions
is termed catabolite repression and is as a result of the
low levels of cAMP that results from an adequate
glucose supply.
This repression is maintained until the glucose supply is
exhausted.
What happens when glucose levels start dropping
in the presence of lactose?
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As the level of glucose in the medium falls, the level of
cAMP increases.

Simultaneously the inducer (allolactose) is also binding
to the lac repressor (since lactose is present).
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The net result is an increase in transcription
from the operon.
The ability of cAMP to activate (increase)
expression from the lac operon results from
an interaction of cAMP with a protein
termed CRP (for cAMP receptor protein).
The protein is also called CAP (for
catabolite activator protein).

The cAMP-CAP complex binds to a region of the lac operon
just upstream of the promoter
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The binding of the cAMP-CRP complex to the lac operon
stimulates RNA polymerase activity 20-to-50-fold.

(Repression of the lac operon is relieved in the
presence of glucose if excess cAMP is added.)
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cAMP is therefore an activator of the lac operon.
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This type of regulation by an activator is positive in
contrast to the negative control exerted by repressors.
trp operon
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The trp operon encodes the genes for the synthesis of tryptophan.
As with all operons, the trp operon consists of the promoter, operator and
the structural genes.
It is also subject to negative control by a repressor
In this system, unlike the lac operon, the gene for the repressor is not
adjacent to the promoter, but rather is located in another part of the E. coli
genome.
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Another difference is that the operator resides entirely within the promoter
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Unlike an inducible system, the repressible operon is usually turned on.
Structure of the trp operon
The operon consists of:
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The operon consists of 5 structural genes that code for
the three enzymes required to convert chorismic acid into
tryptophan’
The operon also contains a gene coding for a short
oligopeptide (trpL) which functions in attenuation
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Operator
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promoter
Gene
Gene Function
P/O
Promoter; operator sequence is found in the promoter
trp L
Leader sequence; containing attenuator (A) sequence
trp E
Gene for anthranilate synthetase subunit
trp D
Gene for anthranilate synthetase subunit
trp C
Gene for glycerolphosphate synthetase
trp B
Gene for tryptophan synthetase subunit
trp A
Gene for tryptophan synthetase subunit
the leader
Negative control of trp operon
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The affinity of the trp repressor for binding the operator
region is enhanced when it binds tryptophan, blocking further
transcription of the operon and, as a result, the synthesis of
the three enzymes will decline.
hence tryptophan is a corepressor.
This means that when tryptophan is absent expression of the
trp operon occurs
the rate of expression of the trp operon is graded in response
to the level of tryptophan in the cell.
Attenuation of the trp operon
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Expression of trp operon is reduced by the
addition of trytophan in trpR mutants.
Further research established that this second level
of tryptophan control involved two components:
1. tRNA, specifically tryptophanyl-tRNA,
tRNATrp, i.e. tRNATrp charged with
tryptophan.
2. the trpL gene
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The attenuator region is composed of sequences
found within the transcribed RNA of the operon
It is involved in controlling transcription from the
operon after RNA polymerase has initiated
synthesis of the proteins.
The leader sequences are located prior to the start
of the coding region for the first gene of the operon
(the trpE gene).
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The leader sequence (trp L) contains tandem
tryptophan codons.
How does this affect transcription of the trp
operon?
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It contains two consecutive trp codons and therefore
serves to measure the tryptophan supply in the cell.
If the supply is good, then the tRNA will be charged
and the leader peptide will be translated without
problem.
If the supply is inadequate, then the tRNA will not be
charged, and translation will stall at the trp codons.
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The trpL mRNA region can adopt a number of different
conformations. It contains several self-complementary
regions which can form a variety of stem-loop structures
Different stem-loops can form:
– Depending on the level of tryptophan in the cell and
hence the level of charged trp-tRNAs
– Depending on the position of ribosomes on the leader
polypeptide and
– Depending on the rate at which they are translated
trp Operon Transcription Under High
Levels of Tryptophan
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Recall that transcription and translation can occur
simultaneously in bacteria.
This means that the ribosome will attach to mRNA
and is able to influence the formation of secondary
structures by the mRNA.
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In the case of the trpL mRNA, when the cellular levels of
tryptophan are high, the levels of the tryptophan tRNA are
also high.
Immediately after transcription, the ribosome follows right
behind RNA polymerase until it is halted by a stop codon.
Translation is quick because of the high levels of tryptophan
tRNA.
This permits formation of the terminator stem-loop which
will cause RNA polymerase to dissociate (recall rho
independent termination of transcription in prokaryotes)
How is the terminator stem-loop
formed?
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Because of the quick translation of domain 1, domain 2
becomes associated with the ribosome complex.
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Then domain 3 binds with domain 4, and transcription is
attenuated because of this stem loop formation.
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The stem loop formed by binding of domains 3 and 4 is
found near a region rich in uracil and acts as the
transcriptional terminator loop (see transcription notes from
unit 3)
Consequently, RNA polymerase is dislodged from the
template.
trp Operon Transcription Under
Low Levels of Tryptophan
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Under low cellular levels of tryptophan, the translation of the short
peptide on domain 1 is slow.
As a result domain 2 does not become associated with the ribosome.
Rather domain 2 of the leader mRNA associates with domain 3 of the
leader mRNA.
This step loop structure is the anti-terminator. Its formation prevents
formation of the terminator
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This structure permits the continued transcription of the
operon. Then the trpE-A genes are translated, and the
biosynthesis of tryptophan occurs
Domain 4 is called the attenuator because its presence is
required to reduce (attenuate) mRNA transcription in the
presence of high levels of tryptophan.

Domain 1 is also an important component of the attenuation
process.
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The section of the leader sequence encodes a 14 amino acid
peptide that has two tryptophan residues.
References
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http://www.biocourse.com/ui/swf/iLab
s/lac_operon.swf
http://faculty.plattsburgh.edu/donald.s
lish/Att-Trp.html
http://highered.mcgrawhill.com/sites/dl/free/0072835125/126
997/animation28.html