BIO353.

01 2021/22-1

Lecture 13: Regulation of Transcription in Prokaryotes (simplified)

Slide 2
The expression of genes needs to be tightly controlled to serve the moment-by-moment need of a cell.
Genes are turned on or off in response to different environmental conditions (e.g. nutrient availability,
temperature, cell density) but also at different stages of the life cycle of the organism or cell. At one
extreme, genes are controlled by simple on/off switches, while others are more precisely tuned to
allow for graded expression to tune the ratio of gene product that is generated from a large number of
genes.

Slides 3 / 4
We have already see that the efficiency with which RNA pol is recruited to the promoter determines
the level of gene expression. In the simplest form, the baseline rate of expression is hard-wrired into
the promoter sequences themselves. Compliance with the consensus sequences for the -10 and -35
elements endows different promoters with different transcriptional activity. Promoters that contain
motifs that are very similar to the consensus sequence are more active than promoters that diverge
from the ideal sequence. Additional sequences that recruit binding factors that interact with the aCTD
of RNA pol further increase the attractiveness of the promoter. This is an example of cooperative
binding/recruitment.

Slide 5
Another mechanism by which additional factors that are recruited to sites near the promoter could
modulate the rate of transcription of a gen is by allosteric interaction with the polymerase. For
instance, the additional factor could stimulate the transition from the closed to the open complex.

Slide 6
Initial insight into how genes are regulated in bacteria comes from the groundbreaking work of Fancois
Jacob and Jacques Monod, who established the lac operon as a well understood genetic switch that is
turned on only under certain nutrient conditions.

Slide 7
In bacteria, genes that code for proteins that function in the same metabolic pathway are often
grouped and expressed as polycistronic mRNAs. The -10 and -35 sites contribute to recruitment of
RNA pol to the promoter but by itself provide little opportunity for dynamic regulation of gene
expression. Rather the expression levels are rigidly encoded by the exact sequence definition.

Slide 8
The operon concept adds possibilities for dynamic regulation of gene expression, i.e. that genes can
be turned on or off under different conditions. Essentially expression is regulated by activators and/or
repressors of gene expression that bind to additional sites surrounding the core promoter of a gene or
operon. Activators bind to activator binding sites, while repressor proteins bind to a site that Jacob and
Monod termed the operator. The whole array of activator binding site, core promoter, and operator is
included in the definition of the operon.

Slide 9
Activators assist cooperatively in bringing RNA pol to the promoter site. They interact with DNA and
RNA pol simultaneously and, thereby, provide additional binding sites. The mode of action can be
either recruitment or allosteric regulation of RNA pol by affecting the ability to undergo transition as
discussed above.

Slide 10
Repressors, on the other hand, somehow interfere with RNA pol binding by blocking sites within the
core promoter or elongation by occupying DNA ahead of RNA pol movement.

Slide 11
The system studied by Jacob and Monod were the genes that allow E. coli to utilize lactose sugars as
an alternative form of energy when their preferred nutrient, glucose, is not available. In a regular
culture supplied with glucose as a nutrient, bacteria will grow exponentially but soon will use up all of
the glucose within the growth medium. As a consequence, bacterial growth will stagnate.

Slide 12
If, however, alternative nutrients, such as lactose, are supplied with the medium, bacteria undergo a
shift and start to utilize lactose as soon as the glucose concentration reaches a certain low level.

Slide 13
To efficiently utilize lactose, E. coli cells need to perform three distinct processes. They need to be
able to transport lactose molecules across the bacterial wall and membrane, they need to be able to
metabolically break down lactose into galactose and glucose, and they need to protect themselves
against potentially harmful substances that can be transported into the cell by the same transporters
that allow for lactose uptake.

Slide 14
The genes encoded by the lac operon code for three distinct proteins that perform these functions. b-
galactosidase, encoded by the lacZ gene enzymatically breaks down the lactose disaccharide, lactose
permease (lacY) transports lactose into the bacterial cells, and the lacA-encoded thiogalactoside
tranacetylase destroys harmful thiogalactosides. All three genes are encoded by a single tricistronic
mRNA controlled by the same operon.

Slide 15
The lac operon contains an activator-binding site, also called a CAP site at which the CAP protein
(catabolite activator protein) binds to DNA. An operator flanking the transcription start site can be
bound by a lac repressor protein lacI. CAP not only binds to DNA at the CAP site but also detects the
levels of glucose in the medium. Only of glucose levels are low, will CAP bind to the lac operon. The
lac repressor has a similar property and binds to the operator only in the absence of lactose.

Slide 16
The lac promoter is a weak promoter. Both the -10 and -35 sites are suboptimal and RNA pol will bind
only weakly in the absence of the CAP activator (not shown here). The lac repressor binds to a site
just downstream of the transcription start (red highlight).

Slide 17
Together, the lac repressor and CAP integrate information and control gene expression at the lac
operon. If both glucose and lactose are available, neither CAP nor lacI bind to the operon. Thus, the
lacZYA genes are only expressed at some basal level that is provided by the -10 and -35 sites in the
weak core promoter. If glucose is present but lactose is absent, the repressor would shut down this
basal level of transcription since nothing can be gained from the expression of the lac genes. Only if
glucose is absent and lactose present, will the operon be activated by binding of CAP and the lack of
repressor binding.

Slide 18
This slide shows the arrangement of DNA sites within the lac operon. The operon actually includes
three operaters, not just one. Operator 1 (O1) is the one that is located just downstream of the TSS,
while O3 is located upstream of the CAP-binding site. The third operator, O2, is further downstream
into the lacZ gene and not shown here. A multitude of mutations exist that change the affinity of the lac
operon for RNA pol. The UV5 lac promoter includes a -10 site that has been corrected to become an
optimal -10 site.

Slide 19
In the absence of additional regulators, changing only two nucleotides in the -10 site increases
transcription about 17-fold. This is similar to activation of the wild type promoter by CAP.

Slide 20
The CAP protein belongs to a class of DNA-binding proteins that are termed helix-turn-helix proteins.
They interact with DNA by inserting an a-helix into the major groove of DNA to access sequence
information without breaking the hydrogen bonds between the DNA strands. A second helix interacts
with the surface of the DNA molecule to properly orient the recognition helix into the groove. The
functional CAP protein is a dimer and each recognition helix interacts with a half-site on the DNA.
Slide 21
CAP is a DNA-binding protein but also a sensor of low glucose levels. Actually, CAP does not sense
glucose directly but rather is sensitive to the small molecule cAMP, which increases in bacterial cells
when energy levels are low. cAMP binds to CAP and induces a conformational change that results in
a rotation of the recognition helices so that they can insert into the major groove of DNA. The distance
between the helices in the cAMP-bound form is 34 Å, which corresponds to one full helical turn of
DNA.

Slide 22
CAP interacts with the aCTD of RNA pol to facilitate its recruitment to the promoter.

Slide 23
Mutations in RNA pol that prevent CAP interaction significantly lower recruitment and expression from
the lac operon.

Slide 24
The lac repressor has a similar structure and also binds as a dimer to two adjacent half-sites around
the TSS.

Slide 25
The lac repressor protein bound to the promoter occupies a region that is largely overlapping with the
footprint of RNA pol on the promoter.

Slide 26
Similar to CAP, lacI does not directly bind to lactose but to allolactose, which is an enzymatic product
of b-galactosidase. Other structural derivatives of allolactose can also activate lacI, such as IPTG.

Slide 27
Substrates bound to lacI also result in a rotation of the recognition helices but this time, the helices are
positioned so that they do not fit into the major grooves when IPTG or allolactose are bound.

Slides 28 / 29 / 30
You may have asked yourself, why the lac operon contains three operators when the one at the TSS
seems to be sufficient to prevent RNA pol binding. Lac repressor dimers can form tetramers with
dimers bound to any of the other operators to force DNA looping. The looping further increases the
strength of repression. The idea here is that the looped DNA is even less accessible to RNA
polymerase. The repressor can only form tetramers but being able to form more than just one loop,
increases its repressive function.

Slide 31
The araBAD operon is another example, which uses similar properties to control the expression of
genes that allow E. coli to use the sugar arabinose as an alternative energy form. The operon consists
of four binding sites for the repressor protein araC, which controls expression of the araBAD genes.
The repressor sites are organized into two operators, O1 and O2, which have low and high affinity for
araC. An additional site araI also binds araC with high or low affinity and functions as a repressor or as
an inducer site, depending on the presence/absence of arabinose. The operon also includes a CAP
site, similar to the lac operon. Expression of araC is controlled from sites that overlap with the sites
that control araBAD expression.

Slide 32
In the absence of arabinose (and presence of glucose), araC binds to the two high affinity sites in araI
and O2 to form a loop between these sites. The araBAD promoter is even less efiicient than the lac
promoter (see slide 16) and the araBAD genes are almost not expressed in the absence of CAP
bound to the operon. The loop between araI and O2 prevents the interaction of CAP with RNA pol.

Slides 33 / 34
When arabinose is available, it binds to araC, which changes its conformation and affinity for the araC-
binding sites. It now more efficiently binds to the two sites in araI but not to O2. Thus, the DNA loop
does not form and CAP can recruit RNA pol to the promoter when glucose is absent.
Slide 35
The operon also controls the levels of the araC repressor by an autoregulation mechanism. If the
concentration of araC increases, it will also bind to the low affinity site in O1 and will prevent further
expression of araC.

Slide 36
Gene expression may be controlled in fundamentally different ways. For instance, the MerR activator
activates gene expression in the presence of mercury ions. Mercury binds to MerR, which binds to the
promoter and twists the DNA. The promoter contains -10 and -35 sites but with a suboptimal spacing
so that they face different sides of the DNA, which makes binding of the sigma factor to both sites
unfavorable. In the twisted form, the -10 and -35 sites rotate relative to each other to face the same
surface of the DNA molecule.

Slide 37
The NtrC binds to DNA when nitrogen levels are low and forms a loop that bends back to interact with
RNA pol to stimulate transition between the closed and open complex.

Slide 38
Another way to differentially control gene expression is through the use / availability of different sigma
factors. While most genes in E. coli are controlled by s70, alternative factors may control the
expression of genes that should be expressed under special circumstances.

Slide 39
These sigma factors differ in the specific sequences that they recognize in the -10 and -35 elements.

Slide 40
On curious example is s32, which control heat-shock genes that are expressed in a protective
response to high heat. The mRNA coding for s32 forms stable intramolecular structures and cannot
be translated to produce the factor at normal temperature. Heat above 40 ºC melts the mRNA, which
now can be translated and s32 can interact with promoters that have -10 and -35 sites that are
recognized by s32 but not s70.

Slide 41
The examples that we looked at so far had in common that they were involved in catabolic processes.
Anabolic processes are different in that they are often controlled by the end product of the pathway.

Slide 42
The trp operon controls expression of a series of gens that are involved in the anabolic pathway that
results in the production of the amino acid tryptophan. These genes are only expressed if tryptophan
levels are low in the bacterial cells, whereas high tryptophan shuts down their synthesis. The trpR
gene codes for the tryptophan repressor, which binds to the promoter only in the presence of the
amino acid, which functions as a co-repressor. However, the repression of the trp operon by trpR is
only modest and does not abolish gene expression completely.

Slides 43 -
Additional mechanisms contribute to a more efficient silencing of the trp operon and depend on a
mechanism that is called attenuation.

(43) The anabolic pathway is controlled by the trpEDCBA genes. However, the sequence between
the promoter in the trp operon and the trpEDCBA cistrons contains two additional sequences,
the trpL leader and an attenuator sequence, that contribute to the attenuation process.
(44) When transcription initiate, the RNA coding for the leader peptide is synthesized first. The
leader sequence includes a short open reading frame that codes for a peptide that includes
two tryptophane molecules.
(45) When tryptophan is low, the leader and the subsequent trpEDCBA genes are expressed. In
the presence of high levels of tryptophan, the RNA polymerase falls off the DNA template at
the attenuator site and the trpEDCBA genes are not transcribed.
(46) Translation of a newly synthesized RNA molecule in bacteria is immediate and starts
eventually before the RNA transcript has finished.
(47) The attenuator sequence can form two alternative secondary structures, one of which
resembles a transcription termination signal (rho-independent termination).
(48) Which loop structure is favored depends on the presence of tryptophan during translation of
the leader peptide. If no tryptophane is available, the ribosome gets stalled during leader
peptide synthesis because it includes two codons instructing the insertion of tryptophane. As a
consequence, a RNA loop forms that does not function as termination signal and transcription
by RNA pol continues into the trpEDCBA genes.
(49) In the presence of sufficient tryptophan, the ribosome continues beyond the UGG codons and
forces the formation of the termination loop. As a result, RNA polymerase stops transcription
at the end of the attenuator sequence.

Slide 50
Another interesting example that sheds some light onto how coordinated programs of gene expression
can be controlled comes from bacteriophages that infect bacterial cells to replicate.

Slide 51
Most phages have a lytic life style, meaning that they infect the bacteria to form new viral particles that
are leased through lysis of the bacterial cells. Proteins encoded by genes on the phage DNA, is
injected during infection, take over the bacterial cells and form the building blocks of viral particles.

Slide 52
The takeover of the host cells is coordinated and timed. The early genes are involved in shutting down
gene expression of bacterial genes, while the middle genes assist replication of the phage genome
and destruction of the host genome (makes sense since only the phage genome should be
replicated). Only during a late phase of the infection cycle are the genes expressed that code for the
proteins that form the head and tail of the viral particles and which lyse the bacterial cells. Each
transition between early, middle, and late genes requires a switch in gene expression.

Slide 53
How this switch is achieved is solved differently in different phages. The SPO1 phage, which infects
Bacillus subtilis, employs alternative sigma factors. The early gens contain promoters that are
recognized by s70 of the bacterial host. They include a gene that codes for s28, which binds to
promoters of the middle genes. Similarly, one of the middle genes is a gene that codes for s33/34 that
brings RNA pol to the promoters of the late genes.

Slide 54
Not all phages are lytic and some have the capacity to integrate into the host genome, where they
remain inactive. The lytic life style only makes sense when sufficient bacteria are around that can be
infected. In times of retarded growth, the phage would run the risk to die out if it causes the lysis of the
host without a chance of infecting neighboring bacteria. The strategy is to become a part of the
bacterial DNA and to replicate passively with the bacteria until environmental conditions change and
become favorable again. The silent phage genome is called a prohage or a lysogen. During this phase
the phage genes are actively repressed by a repressor protein, which is the only phage gene that
remains expressed during the lysogenic phase.

Slide 55
One particularly well understood example is the lambda phage.

Slide 56
The lambda phage genome is a relatively small piece of DNA of 48.5 kb. In addition to the genes that
form the viral particles, control lysis, or assist integration into the host genome, it also contains specific
genes that constitute a molecular switch that controls whether the phage is lytic or a lysogen.

Slide 57
The phage DNA is a linear molecule inside the viral particles but becomes circularized upon entry into
the bacteria through the joining of cohesive ends with help of bacterial proteins.

Slide 58
During lytic growth, lambda also executes a temporal sequence of gene expression. During the early
phase only two genes are expressed, which are called N and cro. Genes required for the replication of
the phage genome are expressed during the middle phase. It is during this time that the phage
decides whether it wants to continue with lytic growth or if it should become a lysogen. Integration into
the host genome requires the expression of an integrase gene (see lecture 7 on site-specific
recombination) that catalyzes recombination at attB/attP sites. Because the fate of the phage has not
been decided yet, both the recombination and replication genes are expressed. The late phase
includes all the genes that are necessary to form new viral particles. Gene expression of the eraly,
middle, and late genes is essentially controlled by three promoters, which are called PL (leftward
promoter), PR (rightward promoter), PR’. You can see that both the early and middle genes are
transcribed from PL and PR. However, transcription terminates after the N and cro genes during the
early phase, whereas it continues into the adjacent genes during the middle phase.

Slide 59
Switch in gene expression from early to middle and middle to late genes is achieved by a new
mechanism, which is called antitermination. Both the early and middle genes are transcribed from PL
and PR but transcription stops early. One of the two early genes is N, which acts as a factor that
prevents transcriptional termination so that the adjacent genes are included in the transcript. The late
genes are all expressed from PR’ but in the absence of Q, only a short transcript is produced that
does not contain any cistron. Q is one of the middle genes and functions as an antiterminator at PR’.

Slide 60
There are two antitermination events that are regulated by N, one at PL and one at PR. The
mechanism is identical, so we only consider PR here. During the erly phase only cro is transcribed
from PR but the sequence between cro and the middle genes contains a sequence that recruits N to
the mRNA (nutR = N utilization right). The nutR site is not the termination signal itself but affects
termination at a site that is transcribed later tR1.

Slide 61
The tR sites form stem loops followed by stretches of transcribed U nucleotides that function as a rho-
independent termination signal. N, when bound to the mRNA, prevents the formation of the stem-loop
and termination of transcription. As a result, RNA pol can continue to transcribe into the middle genes.

Slide 62
Antitermination by Q uses a different mechanism. The DNA immediately downstream of PR’ includes
a sequence that resembles a -10 site, which causes RNA pol to stall. The promoter itself includes a Q-
binding site (QBE).

Slide 63
When Q is transcribed it can bind the QBE and give the RNA pol the required push to continue
between the pause site.

Slide 64
When the virus enters the bacteria, it has two alternatives and needs to make a decision whether it is
more favorable to become lytic or a lysogen. We will see on the enxt slides that the difference
between these two life styles are mutually exclusive (=alternative) gene expression programs on the
phage DNA. When bacteria are healthy and dividing frequently, lysis has advantages because there
are sufficient bacteria around for subsequent infections. On the other hand, when the number of
bacteria is low, it is a better strategy to ‘hitchhike’ on the life cycle of the bacteria and to remain a silent
passenger in the bacterial genome. If, however, the bacteria that harbor the lysogen are about to die,
another switch (= induction) between the lysogenic and lytic form can occur.

Slide 65
To better understand how these alternative gene expression programs are maintained, how the initial
decision is made, and how the switch works, we need to take a closer look at the promoters and
regulators that play a role in controlling these promoters. WE have already seen the left- and rightward
promoters. The leftward promoter PL is irrelevant for the events considered here but the rightward
promoter PR controls expression of the cro gene. Cro stands for ‘ontrol of repressor and other genes’.
In the same region, two additional promoters, PRM and PRE, exist that control expression of the
lambda repressor that is encoded by the cI gene. Here, PRE stands for ‘promoter for repressor
establishment’, while PRM is an abbreviation of ‘promoter for repressor maintenance’. The first one is
important for the initial decision, the second one for the maintenance of the lysogenic state.
Slide 66
Cro is expressed in lytic cells from PR and binds to PRM to repress expression of the lambda
repressor. Alternatively, in lysogens, lambda repressor is expressed from PRM and represses the lytic
genes from PL and PR.

Slide 67
Structurally, lambda repressor is similar to the lac repressor. It has a helix-turn-helix structure, forms
dimers that occupy two half-sites on DNA but the repressor can also form tetramers at different
operators.

Slide 68
There are a total of 6 operators at which the lambda repressor can bind, three of which are located on
the right side, where they overlap with PRM and PR. The novelty here is that lambda repressor can
both be a repressor and an activator of expression, depending on where it binds to the DNA. It is a
repressor for PR at OR1 and prevents expression of lytic genes. It is also a repressor for PRM at OR3,
where it can shut down expression of itself. When bound to OR2, it acts as an activator of the cI gene
and positively controls its expression.

Slide 69
Cro can also bind to the same operators but lambda and cro have different affinity. Lambda binds
effectively at OR1 to prevent expression of cro and other lytic genes, while cro binds strongly to OR3
to prevent cI expression.

Slide 70
This western blot shows the expression levels of cI and cro in the presence of different concentrations
of lambda repressor. Even a moderate amount of repressor prevents cro expression. You can also
see that a small amount of repressor (0.2) stimulates cI expression and that higher concentrations
decrease expression of cI.

Slide 71
In the lysogen when lambda is expressed, it binds strongly first to OR1 and cooperatively to OR2. In
this configuration, lambda also interacts with RNA pol (similar to CAP) and directs it to PRM, which
allows for additional expression of the repressor gene.

Slide 72
In lytic cells, cro is expressed and binds to the high affinity site OR3, which blocks expression of
lambda repressor from cI but allows RNA pol to engage at PR.

Slide 73
The actual conformation is even more complex, since both the right and the left half have three
binding sites each. Thus, lambda can also force the formation of a loop between operators in the left
and right half, which shuts down expression of cI very efficiently.

Slide 74
This agar plate shows what phage-infected bacteria look like. The plate contains an even spread of
bacteria but some spots are more transparent. These spots are called plaques and are sites where
bacteria have been lysed by lambda. However, the plaques are not completely transparent but contain
a low density of bacteria that are resistant to phage infection. Those are lysogens that have acquired
phage immunity. This makes sense because these cells already contain a high concentration of
lambda repressor, which prevents expression of lytic genes.

Slide 75
What the determines at initial infection, whether a cell will become lysogen or not? To simplify things,
cro and downstream genes are expressed in lytic cells, while cI is expressed in lysogens. One of the
genes downstream of cro is cII.

Slide 76
The protein encoded by cII is an activator or PRE, which will turn on expression of lambda repressor.
Once sufficient amount of lambda has accumulated, it will bind to the operators OR1-3 to shut down
expression from PR and to activate its own expression from PRM.

Slide 77
The decision between lytic growth and lysogenic silence now depends on whether cro or cI win the
race.

Slide 78
What tips the balance between these two is cII, which controls how much cI is initially synthesized
from PRE. cII itself responds to the bacterial metabolism. If sufficient energy sources (nutrients) are
available, the cells are rich in proteases, which degrade cII. A lower amount of cII means that less cI is
stimulated and that cro wins to control the lytic life style. If cells are starved, stable cII stimulates more
cI expression, which drives the cells into the lysogenic state.

Slide 79
The remaining question is, how the switch is flipped from the lysogen to become lytic again when
survival of the bacteria is threatened. A main indicator is accumulated DNA damage that, for instance,
occurs when bacteria are exposed to strong UV light.

Slide 80
UV light causes DNA double strand breaks, which induce DNA repair by homologous recombination.
An important component of the pathway is the RecA protein, which allows for strand invasion and
homology search.

Slide 81
RecA also has protease activity and cleaves the LexA gene that acts as a repressor of SOS genes
that contribute to DNA repair but also includes other genes involved in stress responses.

Slide 82
LexA and lambda share some similarity and lambda can also be cleaved by RecA. Thus, in cells in
which DNA is damaged, the concentration of lambda repressor drops below a critical point that is
necessary to maintain the lysogenic state. The SOS genes also include an antirepressor that
contributes to the switch.

End of transcript !!! - (shf, 03.12.2021)