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Chapter 16 Gene regulation in
Prokaryotes
Gene expression is controlled by
regulatory proteins
• Regulatory proteins: positive regulators, or
activators; and negative regulators, or
repressors.
• They are typically DNA-binding proteins
that recognize specific sites at or near the
gene they control.
Many promoters are regulated by activators that help RNA
polymerase bind DNA and by repressors that block that
binding
• At many promoters, when RNA
polymerase does bind to the
promoter, it spontaneously
undergoes a transition to the
open complex and initiates
transcription, called basal level.
• The site on DNA where a
repressor binds is called an
operator.
• Some activators help
polymerase bind the promoters.
This mechanism, called
recruitment, is an examples of
cooperative binding of protein
to DNA.
Some activators work by allostery and regulate
steps after RNA polymerase binding
• Some promoters require activators to stimulate the
transition from closed to open complex.
• Activators that stimulate this kind of promoter
work by triggering a conformation change in
either RNA polymerase or DNA.
• This mechanism is an example of allostery.
• One activator, NtrC, interacts with the RNA
polymerase bound in a closed complex at the
promoter and stimulates transition to the open
complex. It is an example of σ54 holoenzyme
transcription.
Allosteric activation of RNA polymerase
Action at a distance and DNA looping
• NtrC activates a promoter
“from a distance”: its
binding sites are normally
located about 150 bp
upstream of the promoter.
• One way to help bring
distant DNA sites closer
together (and so help
looping) is the binding of
other proteins to sequences
between those sites.
DNA-bending protein
• There are cases in which a protein binds between an
activator binding site and the promoter and helps the
activator interact with polymerase by bending the
DNA
Cooperative binding and allostery
have many roles in gene regulation
• Simple cooperative binding: the activator interacts
simultaneously with DNA and with polymerase
and so recruits the enzyme to the promoter.
• Allostery is not only a mechanism of gene
activation, it is also often the way regulators are
controlled by their specific signals.
• A typical bacterial regulator can adopt two
conformations- in one it can bind DNA, in the
other cannot, depends on the presence of a signal
molecule.
Regulation of transcription initiation:
examples from bacteria
• The lac Operon: It contains three structural genes – genes
that code for proteins : -galactosidase (lacZ),
galactoside permease (lacY), and galactoside
transacetylase (lacA).
• They all are transcribed together on one m RNA, called a
polycistronic message, starting from a single promoter.
The mechanism of Repression
• The lac operator overlaps the promoter, and so repressor bound to
the operator physically prevents RNA polymerase from binding to
the promoter.
• Negative Control of the lac Operon
• Repressor-operator Interactions
Positive Control of the lac Operon
• It is mediated by a factor called catabolite activator
protein (CAP) in conjunction with cyclic AMP, to
stimulate transcription.
• Sensed the lack of glucose, increase of cAMP.
CAP is dimeric and binds to 22 bp operator sequences,
accelerates the initiation of transcription at these promoters.
CAP has separate activating and DNA binding surfaces
CTD
CAP and lac repressor bind DNA using a
common structural motif
• Recognition of specific DNA sequences is achieved
using a conserved region of a helix-turn-helix.
• This domain is composed of two alpha helices, one is
the recognition helix.
Lac repressor binds as a tetramer to two operators: in such case, the
interventing DNA loops out to accommodate the reaction.
The activities of Lac repressor and CAP are
controlled allosterically by their signals
• It is allolactose (rather than lactose itself) that
controls Lac repressor.
• Allolactose binds to Lac repressor and triggers a
change in the shape (conformation) of the protein.
• Glucose lowers the intracellular concentration of a
small molecule, cAMP. This molecule is the
allosteric effector for CAP.
• Only when CAP is complexed with cAMP does
the protein adopt a conformation that binds DNA.
Partial diploid cells show that functional repressors work in trans.
Partial diploid cells show that operators work only in cis.
Alternative  factors direct RNA polymerase
to alternative sets of Promoters
• Heat shock  factor is 32. When E. coli is
subject to heat shock, the amount of this
new  factor increases in the cell, and
displaces  70 from a proportion of RNA
polymerases.
•  54 in the cells is required to transcribe
genes involved in nitrogen metabolism.
• Transcription of phage SPO1 genes in infected B.
subtilis cells proceeds according to a temporal
program in which early genes are transcribed first,
then middle genes, and finally late genes. This
switching is directed by a set of phage-encoded σ
factors that associated with the host core RNA
polymerase and change its specificity from early to
middle to late.
NtrC and MerR: transcriptional activators that
work by allostery rather than by recruitment
• The majority of activators work by recruitment.
• Two exceptions: NtrC and MerR.
• In the case of activators that work by allosteric mechanisms, polymerase
initially binds the promoter in an inactive complex. The activator triggers
an allosteric change in that complex.
• NtrC induces a conformational change in the enzyme that triggers open
complex formation.
NtrC has ATPase activity and works
from DNA sites far from the gene
• NtrC binds to each site as a dimer, and through
protein-protein interactions between the dimers,
binds to the four sites in a highly cooperative
manner
MerR activates transcription by twisting
promoter DNA
• MerR binds to a sequence located between the -10 and -35 regions of the
merT promoter.
• MerR binds to the opposite face of the DNA helix from that bound by RNA
polymerase.
• When MerR binds to Hg2+, the protein undergoes a conformational change
that causes the DNA in the center of the promoter to twist.
• It is an example of altering the conformation of DNA in the vicinity of the
prebound enzyme.
AraC and control of the araBAD operon by
antiactivation
• The promoter of the araBAD
operon from E. coli is
activated in the presence of
arabinose and the absence of
glucose and directs
expression of genes
encoding enzymes required
for arabinose metabolism.
• Activator AraC adopts
different conformations in
the presence or absence of
arabinose.
Examples of gene regulation at steps after
transcription initiation
• In E. coli the five contiguous trp genes encode enzymes
that synthesize the amino acid tryptophan.
• Tryptophan acts as a corepressor, not an inducer.
• When tryptophan is present, it binds to Trp repressor and
induces a conformational change in that protein and
enables it to bind the trp operator.
Amino acid biosynthetic operons are controlled
by premature transcription termination
• Once polymerase has initiated a trp mRNA molecule,
it does not always complete the full transcript.
• The mechanism overcomes the premature
transcription termination is called attenuation.
The case of phage : layers of regulation
• Phage  can replicate in either
of two ways: lytic and
lysogenic.
• A bacterium harboring the
integrated phage DNA is
called a lysogen
• The integrated DNA is called a
prophage.
• The switch from lysogenic to
lytic growth is called
lysogenic induction.
Alternative patterns of gene expression
control lytic and lysogenic growth
•  has a 50-kb genome and some 50 genes.
• Promoters in the right and left regions of phage 
Promoters in the right and left control regions of phage 
PR and PL ( stand for rightward and leftward promoter) are
strong promoters.
PRM ( promoter for repressor maintenance), transcribing only the
cI gene, is a weak promoter and only directs efficient
transcription when an activator is bound just upstream.
Transcription in the  control regions in lytic and lysogenic growth
cI gene encodes  repressor
Regulatory proteins and their binding sites
• The cI gene encodes  repressor, a protein of two domains
joined by a flexible linker region.
• As an activator,  repressor works like CAP, by
recruitment.  repressor’s activating region is in the Nterminal domain of the protein.
• Cro (control of repressor and other things) only represses
transcription.
 and cro can each bind to any one of six operators.
OR1,OR2 and OR3 in the right operators are similar in sequences
but not identical.
The affinities of these various interactions, however, are not all the
same.
 repressor binds to operator sites
cooperatively
 repressor at OR1 helps repressor bind to the
lower affinity site OR2 by cooperative binding.
Repressor and Cro bind in different patterns
to control lytic and lysogenic growth
During lysogeny, PRM is on , while PR and PL are off. This repressor binds to OR1 and
OR2 cooperatively, but leave OR3 open. RNA polymerase binds to PRM,, in a way that
contacts the repressor bound to OR2.
For lytic growth, a single Cro dimer is bound to OR3; this is overlaps PRM
and so Cro represses that promoter.
Lysogenic induction requires proteolytic cleavage
of  repressor
• When a lysogen suffers DNA damage, it induces
the SOS response.
• The initial event in SOS response is the appearance
of a coprotease activity in the RecA protein and
then it stimulates autocleavage of LexA, that
represses genes encoding DNA repair enzymes.
• This causes the repressors to cut themselves in half,
removing them from the  operators and inducing
the lytic cycle.
Another activator, λcII, controls the
decision between lytic and lysogenic
growth upon infection of a new host
The promoter used for establishment of lysogeny is PRE.
Delayed early transcription from PR gives cII mRNA that is
transcribed to CII, which allows RNA polymerase to bind to PRE and
transcribe the cI gene
PRM comes into play after transcription from PRE makes possible
that burst of repressor synthesis that establishes lysogeny.
Growth conditions of E. coli control the
stability of CII protein and thus the
lytic/lysogenic choice
• When the phage infects healthy bacterial
cells, it tends to propagate lytically.
CII is a very unstable protein and is degraded by a specific protease called FtsH.
If growth is good, FstH is very active, then CII is low. In poor growth conditions,
slow degradation of CII leads to lysogeny.
Transcription antitermination in λ development
• N and Q only work on genes that carry particular sequences.
• Five proteins (N, NusA, NusB, NusG and NusE) collaborate bind to
RNA transcribed from DNA containing a nut (for N utilization)
sequence in antitermination in the early operons of .
• Antitermination in the  late region requires Q, which binds to DNA
sequences (QBE) between the –10 and –35 regions of the late gene
promoter (PR’).
N utilization
Cro gene product blocks the
transcription of  repressor CI
N: antiterminator
Extension of transcripts
controlled by the same
promoters. Q: antiterminator