Chapter 5, BIO 3335

REGULATION OF PROKARYOTIC GENE EXPRESSION

The expression of most genes in bacteria is regulated. Gene products are generally not
synthesized if they are not needed. This is really a matter of life and death. Nutrients are
limiting, and organisms that waste time making unnecessary proteins will not survive.
The major forms of control are how much transcript is made (transcriptional control), and
how much is translated (translational control). Most often, control is at the transcriptional
level. There are other forms of control: mRNA degradation, modification of protein activity,
and protein degradation.
TRANSCRIPTIONAL CONTROL
Transcription usually occurs in several steps.
Specific binding of RNA polymerase to DNA.
Strand separation, also called isomerization.
Elongation of RNA, which occurs as a bubble of RNA polymerase between the DNA
strands.
Regulation can occur at any of these steps, but especially the first two.
The structural gene refers to the coding region. Just outside the structural gene is the
regulatory region, which contains sequences involved in control. Regulatory proteins
bind these sequences.
Positive control: Activators can stimulate transcription by helping binding or
isomerization.
Negative control: Repressors usually block the binding of RNA polymerase to DNA.
Regulatory proteins usually exist in two states, active and inactive, which is detemined
by some small molecule (lactose inactivates the Lac repressor).
Regulatory proteins: DNA-binding proteins.
Regulatory proteins make specific contacts with the sequence of DNA. They do so by
specific hydrogen bonds between the protein amino acids and the DNA bases. The
interactions involves the parts of the bases that are not involved in base pairing.
Several types of protein structure allow specific binding to DNA. The point of the
structure is to expose the amino acid residues that interact with DNA. Sometimes it is the
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function of the metabolic effector to cause a conformational change that exposes the
DNA-binding region.
Helix-turn-helix proteins: a glycine in the turn is conserved.
Repressors usually bind as dimers, with each subunit binding to a half site.
Operators sites are B-form DNA.
One of the helices can fit entirely in the major groove of the DNA, where specific
contacts are made.
Zinc fingers: zinc binds to cysteines and histidines, which stabilizes the structure.
Exposed residues contact bases in the major groove.
Leucine zipper proteins: the leucine zipper (patches of hydrophobicity) hold two
subunits together. A basic (positively charged) region contacts DNA.
OPERONS A common strategy for gene expression is the grouping of genes of similar
function near each other. Such genes often form an operon, which is a unit of transcription.
The coding regions for several proteins can be on one mRNA (the mRNA is said to be
polycistronic).
The advantage of an operon structure is that it has one regulatory region for several
genes.
Sometimes genes with common functions are not grouped, but are scattered on the
chromosome. Each such gene may still have a common regulator.
MODELS OF GENE EXPRESSION
The lacZYA operon: Lactose induces expression of the lac operon. Its study led to Nobel
Prize since its study led to ideas about regulatory proteins, regulatory sites, and how the
environment affects gene expression.
LacZ is -galactosidase, which cleaves -galactosides, such as lactose (see structure on p
197). LacY is the lactose permease, a transport protein. LacA is a protein of unknown
function. Loss of either LacZ or LacY prevents growth on lactose as the carbon source.
The lacI gene is adjacent to, but not part of the lacZYA operon (i.e., it is not transcribed
with the lac operon). LacI is a repressor. Its synthesis is constitutive (constant). The
default regulation is repression.
Control features (Figs 5-2 and 5-3):

Expression requires both the absence of glucose, and the presence of lactose.
Lactose is converted to allolactose by the small amount of -gal present in cell (a
minor side reaction). A minor promoter, P2, perhaps provides basal synthesis of galactosidase.
Allolactose binds to LacI, which no longer binds to operator DNA. Allolactose binds
to the allosteric site (other site), and that blocks or causes a conformational change in
the DNA binding site.
Experiments that use the lac promoter often use IPTG to induce. IPTG (isopropylthiogalactoside) is not cleaved, but binds to the repressor. It is called a gratuitous inducer.
Mutations that proved the model:
lacI mutants: lactose not needed for expression. Expression is said to be constitutive.
lacI S mutants: altered inducer binding site. Results in a repressor that always binds
DNA, and therefore cannot be induced.
lacO C mutants: mutated operator site that no longer binds repressor. Like lacI
mutants, these are constitutive.
DNA looping
Weaker repressor binding sites also exist: one within lacI at 100 and the second
within lacZ (the regulated gene) at +400. One repressor tetramer binds both, which
apparently is more stable than binding to a single site.
The OI loop represses initiation, while the OZ loop inhibits RNA synthesis.
When genetics were done (weak sites mutated), the weak sites contribute to repressor
binding, but not as dramatically as at the primary repressor site.
CATABOLITE REPRESSION
The lac operon is only expressed if glucose is not present. Glucose is the preferred
carbon source. The cell does not waste energy making enzymes of lactose metabolism if
glucose is present.
Fig 5-4 shows the effect of glucose addition for cells grown with succinate plus IPTG (an
inducer). Two phases of glucose effect: transient and permanent repression. Cyclic AMP
(cAMP) overcomes both.
The repression is called catabolite repression.
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The enzyme that makes cAMP is regulated: cAMP is low with glucose, and high with
other carbon sources.
The cAMP receptor protein (CRP) binds cAMP, and together they bind to DNA and
activate transcription from P1 and repress P2. CRP bends DNA, which facilitates RNP
binding.
CRP-cAMP controls many operons, some positively, others negatively. This means
that the lac operon has a general regulator that controls several operons, and a specific
regulator just for the lac operon. This pattern of two regulators is common.
Fig 5-5 shows how cAMP synthesis is controlled. A component of the
phosphotransferase system has an interesting regulatory function.
EIIAglc~P stimulates adenylate cyclase activity, and this form of EIIAglc is present
when glucose is not transported into the cell.
EIIAglc inhibits adenylate cyclase, and this form of EIIAglc is present when glucose
transport removes P.
In addition, unphosphorylated EIIAglc inhibits several transport systems, such as
the lactose permease. This is called inducer exclusion.
CRP-cAMP also cooperatively binds with LacI, which increases repression without
lactose.
CRP-dependent transcriptional activation (Fig 5-6).
CRP activates transcription by two different mechanisms. They differ by where CRP
binds to DNA.
In class I promoters, CRP binds at a site centered at 61 bases from the start site.
CRP has a patch called activating region I that contacts the C-terminal domain of the
RNP subunit.
In class II promoters, CRP binds at about 41. RNP binds upstream and contacts AR1,
and downstream and binds AR2. AR2 interacts with the N-terminal domain of the
subunit.
Cra (catabolite repressor/activator) (Fig 5-7).
This regulator controls peripheral pathways.
Cra is displaced by FDP or F6P, and no longer represses.
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THE gal OPERON (Fig 5-8): It is similar to the lac operon: two operator sites, and two
important promoters, control by cAMP. The repressor is different, and the operator sites do
not overlap the RNP binding site. Two repressors must interact, and the HU proteins helps
association
THE ara OPERON (Figs 5-9 and 5-10): Another operon whose products degrade a sugar, in
this case arabinose. It is different because the regulator, the AraC protein, is both a repressor
and an activator. Its mechanism of activation is unusual. It was with this system that looped
DNA for regulation was shown for the first time.
CRP-cAMP together with AraC complexed with arabinose activates both PC and PBAD.
Without arabinose, AraC represses both promoters.
No arabinose, AraC binds O2 and I1, which forms a loop and blocks PBAD.
With arabinose, AraC binds I1 and I2, which also forms a loop, but this loop activates
PBAD. CRP-cAMP may help disrupt the O2-I1 repression loop.
High AraC binds O1 and O2, which represses PC. This mechanism ensures a basal level of
AraC, but not too much.
The CRP site is farther away than normal, and a third activating region is required, AR3.
ATTENUATION MECHANISMS
The trp operon. This was the first studied, and hence the model most often discussed. Two
control mechanisms operate.
The TrpR protein complexed with tryptophan represses. This system responds to
moderate tryptophan starvation.
The second mechanism is attenuation, which responds to more severe starvation.
The basic strategy of attenuation is as follows: with excess tryptophan, transcription
of the trp operon terminates just after it starts, and the operon is not expressed. With
tryptophan starvation, there is no termination, and the operon is expressed. This
strategy, described next, requires the coupling of transcription and translation, which
is not possible in eukaryotes (Figs 5-11 and 5-12).
The region that causes transcription to stop contains a transcription terminator.
The issue is whether the terminator forms.
Between the start of transcription and the first structural gene of the operon, there
is a region called the attenuator. RNA from this region can form a pair of stem
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loops. There are four regions: 1 through 4. RNA by itself forms stem-loops 1-2
and 3-4. Regions 1 and 2 are complementary and can hybridize. Regions 2 and 3
are also complementary, but 2 preferentially binds to region 1.
There is termination if stem-loop 3-4 forms. It is called the attenuator stem-loop.
The presence of tryptophan determines if stem-loop 3-4 forms. RNP can detect
stem-loop 3-4.
The alternate stem-loops form because the RNA is translated and forms a very
short peptide, the leader peptide. This peptide contains two adjacent trp codons.
If tryptophan is not present, then the tRNAtrp is not charged, and ribosomes
stop in region 1. Stem-loop 2-3 (the antiterminator) forms, and there is no
termination.
If tryptophan is present, then the tRNAtrp is charged, and ribosomes stop in
region 2. Stem-loop 3-4 forms, and there is termination.
Notice the use of tRNAs, which bind and sense amino acid levels.
Attenuation is common for other operons of amino acid biosynthesis, for example the his
operon (Fig 5-11).
The pyrBI operon (Fig 5-13). The products of this operon code for the first committed step of
pyrimidine synthesis, aspartate transcarbamoylase. The operon is expressed with low UTP.
There is a U-rich leader mRNA. With low UTP, RNP slows dramatically. With high UTP,
little or no pausing.
There is also a leader peptide. With low UTP, the ribosome catches up with the stalled
RNP, and prevents formation of the terminator step loop. With high UTP, RNP goes past
the pause site, and the terminator forms before the ribosome arrives.
The pyrC strategy (Fig 5-14).
This operon has an amusing variation. There is a 10-fold variation on regulation. The
level of pyrimidines determines the transcription start site. With high pyrimidines,
transcription starts at a C; with low pyrimidines, it starts at a G.
When transcription starts at C, the mRNA has a sufficient region of homology to block
the Shine-Dalgarno sequence (the ribosome binding site), which prevents translation.
Therefore, high C prevents translation.
When transcription starts at G (low pyrimidines), there is not enough of the stem to block
the ribosome binding site.

Other control mechanisms

Flagellar phase variation (Fig 5-16).
Salmonella typhimurium has two types of flagellar proteins. These proteins are the major
antigen of our antigenic response. Antibodies against one are ineffective against the other.
The bacteria can switch.
The basic principle is flipping of the promoter in the DNA so that in one direction it
makes one flagellar protein and a repressor, which blocks synthesis of the other flagellar
gene.
In the other direction, the first flagellar protein and the repressor are not made, so that the
second flagellar protein is made.
Specific recombination proteins mediate the flipping of the DNA.
TRANSLATIONAL REPRESSION: synthesis of ribosomal proteins (Fig 5-17).
The synthesis of ribosomes correlates with growth rate: faster growth requires more
ribosomes.
There are 16 operons, with from 1 to 11 genes that code for the ribosomal proteins. One
protein of a particular operon, when in excess, binds to the mRNA and blocks translation
of those downstream from the repressor site.
These proteins also bind rRNA, so they block translation when there is not enough
rRNA. This prevents synthesis of excess proteins.
The regulatory ribosomal proteins bind to structures of the mRNA that resemble the
structures in rRNA (Fig 5-18).
Posttranscriptional control (Fig 5-19)
The CsrA-CsrB system controls glycogen synthesis and several glycolytic genes in the
opposite direction as Cra.
CsrB is an RNA with 18 repeats of a certain sequence.
It is thought that CsrA binds to the repeats.
In stationary phase, when growth stops, CsrB synthesis goes up. This titrates CsrA,
which binds to mRNA and facilitates their degradation. The effect is more gene
expression.
GLOBAL REGULATORY NETWORKS
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The cell changes several genes in response to certain environments: shifts from rich to
minimal media, carbon/energy limitation, nitrogen limitation, anaerobic conditions, high
temp, heat, low pH, etc. Global regulators control the expression of a network of genes that
permit response to these environments.
A regulon is a group of genes controlled by a single regulator.
A modulon is a group of operons and regulons under one regulator. They are not
necessarily expressed at the same time, such as the CRP modulon.
A stimulon is a group of genes that respond to a common stimulus. A small molecule may
signal the environment, and is called an alarmone.
Two-component regulatory systems: sensing and responding to changing environments (Fig
5-20)
The first component, a sensor kinase, is usually a membrane protein that senses an
environmental factor. It contains a sensor domain (usually membrane bound) and a
transmitter domain (in the cytoplasm). This results in autophosphorylation on a histidine
residue. The activated phosphate is transferred to the next protein.
The second protein is a cytoplasmic protein, often called the response regulator. It
contains receiver and output domains. The phosphate from histidine of the sensor kinase
is transferred to an aspartate of the receiver domain. Response regulators can be
regulators of transcription, but not always.
The duration of the response is controlled by changes in the rates of phosphorylation
and dephosphorylation. The latter is controlled by the stability of phosphorylation
and/or phosphatases.
The structures of these proteins can be complex, and one protein may contain domains
from both components (Fig 5-21).
A hybrid sensor kinase can transfer the phosphate to several residues.
The response regulators are divided into four classes.
E. coli has 32 two-component regulatory systems. They do not send signals to each other,
but are very specific.
Two-component systems control the responses to the following: changes is osmolarity,
phosphate limitation, nitrogen limitation, and chemoattractants and repellents.
Control of nitrogen assimilation and fixation

The pathways of nitrogen assimilation have been discussed previously.

The GS-glutamate synthase pathway is used for high energy and low ammonia.
The GDH pathway for low energy and high ammonia.
Low nitrogen has several effects on enzymes:
it removes a modification of GS, which increases its specific activity and alters its
allosteric properties,
it increases synthesis of GS and other enzymes that assimilate nitrogen from the
environment.
A single regulatory protein, PII, controls both.
Low glutamine signals nitrogen limitation. (The book makes it somewhat more
complicated, and discusses -ketoglutarate also.)
Control of GS activity (Fig 5-22). Low glutamine allosterically affects a set of
regulatory proteins (UTase/UR and PII) which control an enzyme that adenylylates
GS.
With low glutamine, there is no adenylylation. GS has 12 subunits, and each
subunit can be adenylylated. Without adenylylation, each subunit is active.
With high glutamine, these proteins result in adenylylation of GS. This
modification inactivates that modified subunit, and therefore GS is less active.
Control of GS synthesis (Fig 5-23).
Low glutamine, through the same two regulatory proteins, interacts with a sensor
kinase, NRII, which phosphorylates a response regulator, NRI. NRI~P activates the
GS operon and other operons that facilitate the acquisition of nitrogen from the
environment (such as transport systems).
High glutamine results in the shutoff of these genes since NRI is
dephosphorylated.
Unusual features of gene activation.
NRI~P activates RNA polymerase complexed with 54, instead of the major
subunit, 70. is the specificity factor for binding to DNA, and this factor
recognizes a unique sequence.

The activator binding sites can be moved over 1000 bases further away, and still
activate transcription via a long-range protein-protein interaction, like eukaryotic
enhancer binding proteins.
Nitrogenase. In Klebsiella pneumonia which is related to E. coli, this system, called
the Ntr system, also controls nitrogenase synthesis.
NRI~P activates nifLA expression, and NifA activates the nitrogenase genes.
The NifL protein senses oxygen and ammonia, and inhibits NifA activity.
Quorum sensing.
Vibrio fisheri is a free-living organism that can colonize the light organs of squid. At high
cell density, the baceria emit light.
The genes responsible are shown in Figs 5-26 and 5-27. At low cell densities, an
autoinducer is made, an acyl homoserine lactone. With increasing cell densities, the
AHL accumulates and activates the expression of genes required for bioluminescense.
The AHL signal senses cell density.
Other bacteria can have several signals, such as AI-1 and AI-2. This may allow
signaling with other organisms.
The S regulon
When E. coli and S. typhimurium enter stationary phase, S level increases. S also
increases with a variety of sudden stresses, such as low pH or high osmolarity. S then
activates several genes to handle the stress.
S level increases because of increased translation and decreased proteolysis.
The protease responsible is ClpXP.
The RssB protein helps ClpXP bind S. Phosphorylation of RssB is required for this
interaction. The agent that phosphorylates RssB is not known, so the agent that
responds to stress is not known.

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