Control of gene expression

PBT/BI/BN/PMST
• Transcriptional regulation in prokaryotes;
Inducible & repressible system,+ & -ve
regulation; Operon concept, structure of
operon, Lac, Trp, Ara operon, Catabolite
repression, Atteunation.
Control of gene expression
1. transcriptional control,
2. splicing and processing of RNA transcripts (RNA
processing control),
3. selecting which completed mRNAs are exported
from the nucleus to the cytosol and determining
where in the cytosol they are localized (RNA
transport and localization control),
4. translational control,
5. selectively destabilizing certain mRNA molecules in
the cytoplasm (mRNA degradation control), or
6. selectively activating, inactivating, degrading, or
localizing specific protein molecules (protein
activity control)
• For most genes, transcriptional controls are paramount
– Because only transcriptional control ensures that the
cell will not synthesize superfluous intermediates.
Switching genes on and off
• one strategy bacteria employ to control
the expression of their genes:
• by grouping functionally related genes
together so they can be regulated
together easily.
• Such a group of contiguous, coordinately
controlled genes is called an operon.
• Operons are commonly found in
prokaryotes but not in eukaryotes
• Discovered by Jacob and Monod in 1940
• Named the operon concept
• They distinguished between two types of sequences in
DNA:
– sequences that code for trans-acting products (usually
proteins) and cis-acting DNA sequences.
• Gene activity is regulated by the specific interactions of
the trans-acting products with the cis-acting sequences
• A gene is a sequence of DNA that codes for a diffusible
product, either RNA or a protein.
• The crucial feature is that the product diffuses away from
its site of synthesis to act elsewhere.
• Any gene product that is free to diffuse to find its target
is described as trans-acting.
• The description cis-acting applies to any sequence of DNA
that functions exclusively as a DNA sequence, affecting
only the DNA to which it is physically linked.
• A structural gene is simply any gene that
codes for a protein (or RNA) product.
• Protein structural genes represent structural
proteins, enzymes with catalytic activities,
and regulatory proteins.
• One type of structural gene is a regulator
gene
– a gene that codes for a protein or an RNA
involved in regulating the expression of
other genes
• A regulator gene codes for a protein that controls
transcription by binding to particular site(s) on
DNA.
• This interaction can regulate a target gene in
either a positive manner (turns the gene on) or a
negative manner (turns the gene off).
• The sites on DNA are usually (but not exclusively)
located just upstream of the target gene.
• The sequences that mark the beginning and end of
the transcription unit—the promoter and
terminator—are examples of cis-acting sites.
• A classic mode of transcription control in bacteria is
negative control:
– A repressor protein prevents a gene from being
expressed.
– In absence of a repressor the gene is expressed.
– Close to the promoter is another cis-acting site called
the operator, which is the binding site for the
repressor protein.
– When the repressor binds to the operator, RNA
polymerase is prevented from initiating transcription,
and gene expression is therefore turned off.
• An alternative mode of control is positive control.
– This is used in bacteria (probably) with about
equal frequency to negative control, and it is the
most common mode of control in eukaryotes.
– A transcription factor is required to assist RNA
polymerase in initiating at the promoter.
– in the absence of the positive regulator the gene
is inactive: RNA polymerase cannot by itself
initiate transcription at the promoter.
Induction and Repression
• Bacteria need to respond swiftly to changes in their environment.
• Fluctuations in the supply of nutrients can occur at any time, and
survival requires switching from one substrate to another.
• a bacterium avoids synthesizing the enzymes of a pathway in the
absence of the substrate
• The synthesis of enzymes in response to the appearance of a
specific substrate is called induction and the gene is an inducible
gene.
• The opposite of induction is repression, where the repressible
gene is controlled by the amount of the product made by the
enzyme.
– For example, Escherichia coli synthesizes trp through trp
synthetase and four other enzymes.
– If, trp is provided in the medium of growth, production of
enzyme is immediately halted allowing bacterium to avoid
unnecessary synthetic activities.
• Small molecules that cause the production of
enzymes that are able to metabolize them (or
their analogues) are called inducers.
• Those that prevent the production of enzymes
that are able to synthesize them are called
corepressors.
• Typically four different patterns of gene
regulation:
– negative inducible,
– negative repressible,
– positive inducible, and
– positive repressible
• Genes coding for proteins that function in the
same pathway may be located adjacent to one
another and controlled as a single unit that is
transcribed into a polycistronic mRNA
• Typically contains a regulatory gene upstream
Lac Operon

• The lac operon occupies ~6,000 bp of DNA.

• At the left the lacI gene has its own promoter
and terminator.
• The end of the lacI region is adjacent to the
lacZYA promoter, P. Its operator, O, occupies
the first 26 bp of the transcription unit.
• The long lacZ gene starts at base 39 and is
followed by the lacY and lacA genes and a
terminator.
• The protein products enable cells to take up and
metabolize β-galactoside sugars, such as lactose.
• The roles of the three structural genes are as
follows:
– lacZ codes for the enzyme β-
galactosidase, whose active form is a
tetramer of approximately 500 kD.
• The enzyme breaks the complex β-galactoside
into its component sugars (glu and gal).
• also produces an important by-product, β-1,6-
allolactose, with a role in regulation.
– lacY codes for the β-galactoside
permease, a 30-kDmembrane-bound
protein constituent of the transport
system.
• This transports β-galactosides into the cell.
– lacA codes for β-galactoside
transacetylase
• transfers an acetyl group from acetyl-CoA to
β-galactosides.
• For the lac system the natural inducer is not
lactose, but rather a by-product of the LacZ
enzyme, allolactose.
• Allolactose is also a substrate of the LacZ
enzyme, so it does not persist in the cell.
• Some inducers resemble the natural inducers
of the lac operon but cannot be metabolized
by the enzyme.
• The best example of this is
isopropylthiogalactoside (IPTG) and is not
metabolized by β-galactosidase; even so, it is
a very efficient inducer of the lac genes.
• Molecules that induce enzyme synthesis but
are not metabolized are called gratuitous
inducers.
• The separate component that
represses the lac operon is the
lac repressor protein, which is
encoded by the lacI gene.
• The lac repressor protein is
induced by allolactose and
IPTG to allow expression of
lacZYA.
• The LacZ enzyme (β-
galactosidase) utilizes
allolactose and lactose as
substrates.
• lacI is not induced by lactose,
and the LacZ enzyme does not
metabolize IPTG.
• The crucial features of the control circuit reside
in the dual properties of the repressor:
– It can prevent transcription, and it can
recognize the small-molecule inducer.
• The repressor has two types of binding site: one
type for the operator DNA and one type for the
inducer.
• When the inducer binds at its site, it changes the
structure of the protein in such a way as to
influence the activity of the operator-binding
site.
• The ability of one site in the protein to control
the activity of another is called allosteric
control.
Lac Repressor
• The DNA-binding domain
occupies residues 1–59.
• contains two α-helices
separated by a turn (helix-
turn-helix)
• the two α-helices fit into
the major groove of DNA,
where they make contacts
with specific bases
• This region is connected by
a hinge sequence to the
main body of the protein.
• On binding to DNA, the
hinge forms a small α- helix
• when repressor not bound
to DNA this region is
disordered.
• The HTH and hinge are referred to as the
headpiece.
• The remainder of the protein is called the core.
• The bulk of the core consists of two
interconnected regions with similar structures
(core subdomains 1 and 2).
• Each has a six-stranded parallel β- sheet
sandwiched between two α-helices on either
side.
• inducer binds in a cleft between the two regions.
• Two monomer core domains can associate to
form a dimeric version of LacI.
• Dimeric LacI tightly binds operator DNA
because it recognizes both halves of the
operator sequence, which is an inverted repeat
• The C-terminus of the monomer contains
an α-helix with two leucine heptad
repeats. This is the tetramerization
domain.
• The tetramerization helices of four
monomers associate to maintain the
tetrameric structure.
• It consists, in effect, of two dimers.
• The body of the dimer contains an
interface between the subdomains of the
two core monomers and two clefts in
which two inducers bind (top).
• The C terminal regions of each monomer
protrude as helices.
• Together, the two dimers form a
tetramer (center) that is held together
by a C-terminal bundle of four helices.
Catabolite repression
• The E. coli lac operon is negative inducible.
• Transcription is turned on by the presence of lactose
by removing the lac repressor.
• This operon cannot be turned on by lactose if the
bacterium has a sufficient supply of glucose.
• The rationale for this is that glucose is a better
energy source than lactose, so there is no need to
turn on the operon if there is glucose available.
• This is called catabolite repression and is exerted
through a second messenger called cyclic AMP
(cAMP) and the positive regulator protein called the
catabolite repressor protein (CRP) or cAMP receptor
protein or catabolite activator protein, or CAP).
• The lac operon is therefore under dual control.
• CRP is a dimer of two identical subunits
of 22.5 kD, which can be allosterically
activated by a single molecule of cAMP.
• A CRP monomer contains a DNA-binding
region and a transcription activating
region.
• cAMP binding alters the structure of CRP
to change the DNA-binding domain from
one that binds all DNA weakly to strong,
sequence-specific DNA binding.
• A CRP dimer binds to a site of about 22
bp at a responsive promoter.
• The binding sites include variations of the
5-bp consensus sequence
• CRP binds most strongly to sites that
contain two (inverted) versions of the
pentamer, because this enables both
subunits of the dimer to bind to the DNA.
• CRP introduces a large bend when it binds DNA.
• In the lac promoter, this point lies at the center of dyad
symmetry.
• The bend is quite severe, greater than 90°causing a dramatic
change in the organization of the DNA ds
• The mechanism of bending is to introduce a sharp kink within
the TGTGA consensus sequence.
• When there are inverted repeats of the consensus, the two
kinks ineach copy present in a palindrome cause the overall
90° bend.
• It is possible that the bend has some direct effect upon
transcription, but it could be the case that it is needed
simply to allow CRP to contact RNA polymerase at the
promoter
Trp Operon
• The trp operon is a operon that encodes for
the components for the production of
tryptophan
• This trp operon is seen in many bacteria, but
first characterized in E.coli
• The trp operon is called Repressive operon
• Operon has promoter(P), operator(O),
leader(trp L) and structural genes.
• Trp operon consists of 5 structural genes.
• Each gene is responsible for production of
different enzymes.
• trpE : Anthranilate Synthase Component I
• —
trpD : Anthranilate Synthase Component II
• —
trpC : N- (5’-Phosphoribosyl)-Anthranilate
Isomerase and Indole 3-Glycerol Synthase.
• —
trpB : Tryptophan Synthase β subunit
• —
trpA : Tryptophan Synthase α subunit
• —
Together these enzymes catalyze formation
of tryptophan from chorismate.
Mechanism of trp repression
Absence of trp
 Presence of trp

• Trp helps the trp repressor

bind to its operator.
• In absence of trp, the trp
repressor exists as an inactive
protein called aporepressor.

• When the aporepressor binds tryptophan, it changes to a

conformation with a much higher affinity for the trp operator
• The combination of apo repressor plus tryptophan is the trp
repressor; therefore, tryptophan is called a corepressor.
• When the cellular concentration of tryptophan is high, plenty of
corepressor is available to bind and form the active trp
repressor. Thus, the operon is repressed.
• When the tryptophan level in the cell falls, the aminoacid
dissociates from the aporepressor, causing it to shift back to
the inactive conformation; the repressor–operator complex is
thus broken, and the operon is derepressed
Mechanism of trp attenuation
• Second level of regulation
• the trp leader and the trp
attenuator, in between the
operator and the first gene,
trpE.
• The leader–attenuator purpose
is to attenuate, or weaken,
transcription of the operon
when tryptophan is relatively
abundant.
• The attenuator operates by
causing premature termination
of transcription.
– 90% chance of terminating
in the attenuator region
• The reason for premature
termination is that the
attenuator contains a
transcription stop signal
(terminator): an inverted repeat
followed by a string of eight A–T
pairs in a row.
• Because of the inverted repeat,
the transcript of this region
would tend to engage in intra-
molecular base pairing, forming a
―hairpin‖.
• A hairpin followed by a string of
U’s in a transcript destabilizes
the binding between the
transcript and the DNA and thus
causes termination.
Ara Operon

• two ara operators exist: araO1 and araO2.

• The former regulates transcription of a control gene
called araC
• The other operator is located far upstream of the
promoter it controls (PBAD), between positions 2265 and
2294, yet it still governs transcription.
• Second, the CAP-binding site is about 200 bp upstream
of the ara promoter, yet CAP can still stimulate
transcription.
• Third, the operon has another system of negative
regulation, mediated by the AraC protein
• (a) When AraC protein is depleted, the
araC gene is transcribed from its own
promoter.
• (b) When arabinose levels are low and
glucose levels high, AraC protein binds
to both araI and ara02 and brings
these sites together to form a DNA
loop.
• The operon is repressed in this
state. AraC protein also binds to ara0~,
repressing further synthesis of AraC.

• (c) When arabinose is present and

glucose concentration is low, AraC
protein binds arabinose and changes
conformation to become an activator.
The DNA loop is opened, and the AraC
protein acts in concert with CAP-cAmp
to facilitate transcription.