Why does Regulation at the Level of Transcription is Important?

● Transcription initiation is the most energetically efficient step to regulate.

● Regulation at this first step is easier to do, as it is the only a single copy of a gene.

● However, regulation at later stages also have some advantages:

o It allows for more inputs: if a gene is regulated at more than one step, more
signals can modulate its expression, or the same signals can do so even more
effectively.
o Regulation at steps later than transcription initiation can reduce the response
time.
Repressors and Activators:

● At many promoters, in the absence of regulatory proteins, RNA polymerase binds only

weakly; however, when occasionally binds, in the absence of both activator and
repressor, RNA polymerase initiates a low-level (basal level) of constitutive
expression by undergoing a transition to the open complex.
o Binding of the repressor to the operator sequence blocks binding of RNA
polymerase and so inhibits transcription. To activate transcription from the
promoter, an activator can just help the polymerase bind the promoter.
o The activator uses one surface to bind to a site on the DNA near the promoter;
with another surface, the activator simultaneously interacts with RNA
polymerase, bringing the enzyme to the promoter. This mechanism, often
called recruitment, is an example of cooperative binding of proteins to DNA.
The interactions between the activator and polymerase, and between activator
and DNA, serve merely “adhesive” roles: the enzyme is active and the
activator simply brings it to the nearby promoter. Once there, it spontaneously
isomerizes to the open complex and initiates transcription.

● Not all promoters are limited in the same way. In some promoters, RNA polymerase

binds efficiently unaided and forms a stable closed complex. But that closed complex
does not spontaneously undergo transition to the open complex. At this promoter, an
 activator must stimulate the transition from a closed to open complex, since that
transition is the rate-limiting step.
o Activators that stimulate this kind of promoter work by triggering a
conformational change in either RNA polymerase or DNA; that is, they
interact with the stable closed complex and induce a conformational change
that causes transition to the open complex. This mechanism is an example of
allostery.

● Some proteins interact with each other even when bound to sites well separated on

the DNA. To accommodate this interaction, the DNA between the sites loops out,
bringing the sites into proximity with one another.
o Distant DNA sites can be brought closer together to help loop formation. In
bacteria, for example, there are cases in which a protein (DNA-bending
protein) binds between an activator-binding site and the promoter and helps
the activator interact with polymerase by bending the DNA in a favorable
direction. There are also cases where such a protein hinders loop formation
and activation by bending the DNA in an unfavorable direction. Such
“architectural” proteins facilitate (or hinder) interactions between proteins in
other processes as well.

EXAMPLES FROM PROKARYOTES

Lactose Metabolism in E. coli:

● The three lac genes—lacZ, lacY, and lacA—are arranged adjacently on the E. coli

genome and are together called the lac operon. The lac promoter, located at the 5’
end of lacZ, directs transcription of all three genes as a single mRNA (called a
polycistronic message because it includes more than one gene)
o The lacZ gene encodes the enzyme β-galactosidase, which cleaves the sugar
lactose into galactose and glucose.
o The lacY gene encodes the lactose permease, a protein that inserts into the cell
membrane and transports lactose into the cell.
 o The lacA gene encodes thiogalactoside transacetylase, which rids the cell of
toxic thiogalactosides that also get transported in by lacY.
o These genes are expressed at high levels only when lactose is available,
and glucose—the preferred energy source—is not.

● Two regulatory proteins are involved: one is an activator called CAP, and the other is

a repressor called the Lac repressor. The Lac repressor is encoded by the lacI gene,
which is located near the other lac genes, but transcribed from its own (constitutively
expressed) promoter. The name CAP stands for catabolite activator protein, but this
activator is also known as CRP (cAMP receptor protein). The gene encoding CAP is
located elsewhere on the bacterial chromosome, not linked to the lac genes.

● Each of these regulatory proteins responds to one environmental signal and

communicates it to the lac genes.

o Lac repressor can bind DNA and repress transcription only in the absence of
lactose. In the presence of that sugar, the repressor is inactive and the genes
derepressed (expressed).
o CAP can bind DNA and activate the lac genes only in the absence of glucose.

● The site bound by the Lac repressor is called the lac operator. This 21-bp sequence is

twofold symmetric and is recognized by two subunits of Lac repressor, one binding
to each half-site. The lac operator overlaps the promoter, and so the repressor bound
to the operator physically prevents RNA polymerase from binding to the promoter and
thus initiating RNA synthesis.

● RNA polymerase binds the lac promoter poorly in the absence of CAP, even when

there is no functional repressor present. This is because the sequence of the –35 region
of the lac promoter is not optimal for its binding, and the promoter lacks an UP-
element. This is typical of promoters that are controlled by activators.

● In a nutshell, CAP binds as a dimer to a site similar in length to that of the lac

operator, but different in sequence. This site is located some 60 bp upstream of the
start site of transcription. When CAP binds to that site, the activator helps polymerase
bind to the promoter by interacting with the enzyme and recruiting it to the promoter.
This cooperative binding stabilizes the binding of polymerase to the promoter.
● CAP is recognized by the CTDs of the α-subunits. The αCTDs also contact DNA,

adjacent to the CAP site, when interacting with CAP.

o This site is revealed by mutant forms of polymerase that can transcribe most
genes normally, but cannot be activated by CAP at the lac genes. These
mutants have amino acid substitutions in the carboxy terminal domain
(CTD) of the α subunit of RNA polymerase. At the lac promoter, where
there is no UP-element, αCTD binds to CAP and adjacent DNA instead.

● In the typical case, the protein binds as a homodimer to a site that is an inverted

repeat (or near repeat). One monomer binds each half-site, with the axis of symmetry
of the dimer lying over that of the binding site. Recognition of specific DNA
sequences is achieved using a conserved region of secondary structure called a helix-
turn-helix. This motif is composed of two α-helices, one of which—the recognition
helix— fits into the major groove of the DNA. The second helix of the helix-turn-
helix motif sits across the major groove and makes contact with the DNA backbone,
ensuring proper presentation of the recognition helix and at the same time adding
binding energy to the overall protein–DNA interaction.

● This description is essentially true not only for CAP and the Lac repressor, but for

many other bacterial regulators as well, including bacteriophage λ repressor. Despite

this, there are differences
o Lac repressor binds as a tetramer, not a dimer. Nevertheless, each operator is
contacted by only two of these subunits. Thus, the different oligomeric form
does not alter the mechanism of DNA recognition. The other two monomers
within the tetramer can bind one of two other lac operators, located 400 bp
downstream and 90 bp upstream of the primary operator. In such cases, the
intervening DNA loops out to accommodate the reaction.
o In some cases, other regions of the protein, outside the helix-turn helix motif,
also interact with the DNA. The λ repressor, for example, makes additional
contacts using amino-terminal arms. These reach around the DNA and
interact with the minor groove on the back face of the helix.
o In many cases, binding of the protein does not alter the structure of the DNA.
In some cases, however, various distortions are seen in the protein–DNA
complex. For example, CAP induces a dramatic bend in the DNA, partially
 wrapping it around the protein. This is caused by other regions of the protein,
outside the helix-turn-helix motif, interacting with sequences outside the DNA
segment recognized by the helix-turn helix motif. In other cases, binding
results in twisting of the DNA site.

● When lactose enters the cell, it is converted to allolactose. It is allolactose (rather than

lactose itself) that controls the Lac repressor which is catalyzed by β-galactosidase;
because, expression of the lac genes is leaky: even when they are repressed, an
occasional transcript gets made.
o Allolactose binds to the Lac repressor and triggers a change in the shape
(conformation) of that protein.) Once allolactose has altered the shape of the
repressor, the protein can no longer bind DNA, and so the lac genes are no
longer repressed.
o CAP activity is regulated in a similar manner. 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.

● As with the lac genes, the gal genes are only expressed when their substrate sugar, in

this case galactose, is present, and the preferred energy source, glucose, is absent. the
activator of the gal genes is again CAP. Thus, a regulator (CAP) works together with
different repressors at different genes. This is an example of combinatorial control.
In fact, CAP acts at more than 100 genes in E. coli, working with an array of partners.
When the same signal controls multiple genes, it is typically communicated to each of
those genes by the same regulatory protein.

Alternative σ-factors:

● The lac promoter is recognized by RNA polymerase bearing the σ70 subunit but E.

coli encodes several other subunits that can replace σ70 under certain circumstances
and direct the polymerase to alternative promoters.
o One of these alternatives is the heat shock σ factor, σ32. The level of σ32 is
increased by two mechanisms: first, its translation is stimulated, that is, its
 mRNA is translated with greater efficiency after heat shock than it was before;
and second, the protein is transiently stabilized.
o Another example of an alternative s factor, σ 54, is considered in the next
section. σ54 is associated with a small fraction of the polymerase molecules in
the cell and directs that enzyme to genes involved in nitrogen metabolism.
o The bacterial phage SPO1 uses three s factors in succession to regulate
expression of its genome. This ensures that viral genes are expressed in the
order in which they are needed.

Transcriptional Activators That Work by Allostery:

● NtrC controls expression of genes involved in nitrogen metabolism by inducing a

conformational change in a pre-bound RNA polymerase, triggering transition to the

open complex. MerR controls expression of a gene involved in mercury resistance.
MerR also acts on an inactive RNA polymerase–promoter complex, but in this case,
the allosteric effect of the activator is on the DNA, rather than on the polymerase.

● NtrC has separate activating and DNA-binding domains and binds DNA only in the

presence of a specific signal. In the case of NtrC, this signal is low nitrogen levels.
Under these conditions, NtrC is phosphorylated by a kinase, NtrB, and, as a result,
undergoes a conformational change that reveals the activator’s DNA-binding
domain. Once active, NtrC binds four sites located approximately 150 bp upstream of
the promoter (e.g., that of the glnA gene). NtrC binds to each of its sites as a dimer
and, through protein–protein interactions between the dimers, binds to the four
sites in a highly cooperative manner.
o The form of RNA polymerase that transcribes the glnA gene contains the σ54
subunit. Once active, NtrC (bound to its sites upstream) interacts directly with
σ54.
 o NtrC itself has an enzymatic activity—it is an ATPase. This activity provides
the energy needed to induce a conformational change in polymerase.
o At some genes controlled by NtrC, there is a binding site for another protein,
called IHF, located between the NtrC-binding sites and the promoter. Upon
binding, IHF bends DNA.

● When bound to a single DNA-binding site, in the presence of mercury, MerR

activates the merT gene. MerR binds to a sequence located between the –10 and –35
regions of the merT promoter (this gene is transcribed by σ 70-containing polymerase).
MerR binds on the opposite face of the DNA helix from that bound by RNA
polymerase, and so polymerase can (and does) bind to the promoter at the same time
as MerR.
o The merT promoter is unusual. The distance between the –10 and –35 elements
is 19 bp instead of the 15–17 bp typically found in an efficient σ 70 promoter.
When MerR binds Hg+2, however, the protein undergoes a conformational
change that causes the DNA in the center of the promoter to twist. This
structural distortion restores the disposition of the –10 and –35 regions, which
efficiently initiates the transcription.
o In this example, the activator does not interact with RNA polymerase to
activate transcription, but instead alters the conformation of the DNA in the
vicinity of the prebound enzyme.

Holding RNA Polymerase:

● Some repressors work from binding sites that do not overlap the promoter. These

repressors do not block polymerase binding, but instead they bind to sites beside a
promoter, interact with polymerase bound at that promoter, and inhibit initiation.
o In the absence of galactose, Gal repressor keeps the genes off. In this case, the
repressor interacts with the polymerase in a manner that inhibits transition
from the closed to open complex.
o Another example is provided by the P4 protein from a bacteriophage (f29) that
grows on the bacterium B. subtilis. This regulator binds to a site adjacent to
one promoter—a weak promoter called PA3—and, by interacting with
 polymerase, serves as an activator. But this activator also binds at another
promoter, a strong promoter called PA2c. Here, it makes the same contact with
polymerase as at the weak promoter, but the result is repression. It seems that
whereas in the former case, the extra binding energy helps recruit polymerase
and hence activates the gene, in the latter case, the overall binding energy—
provided by the strong interactions between the polymerase and the promoter
and the additional interaction provided by the activator— is so strong that the
polymerase is unable to escape the promoter.

Control of araBAD Operon:

● When arabinose is present, AraC binds that sugar and adopts a configuration that

allows it to bind DNA as a dimer to the adjacent half-sites, araI1 and araI2. Just
upstream of these is a CAP site: in the absence of glucose, CAP binds here and helps
activation.
o In the absence of arabinose, the araBAD genes are not expressed. This is
because when not bound to arabinose, AraC adopts a different conformation
and binds DNA in a different way: one monomer still binds the araI1 site, but
the other monomer binds a distant half-site called araO2.
o When AraC binds in this fashion, the DNA between the two sites forms a loop.
changeIn addition, when bound in this way, there is no monomer of AraC at
araI2, and as this is the position from which activation of araBAD promoter is
mediated, there is no activation in this configuration.
o The promoter is often used in expression vectors, fusing a gene to the araBAD
promoter allows expression of the gene to be controlled by arabinose alone.

Trp-operon:
● It regulates one of the anabolic pathways by presence of the tryptophan amino acid.

When the tryptophan level is high enough, it allosterically binds to repressor, which is
a 70-fold, relatively weak, helix-turn-helix repressor. Before binding to tryptophan, it

exists as aporepressor.
o After RNA polymerase binds to the promoter it starts to transcribe the leader
attenuator and trpEDCBA. Because the leader protein has two tryptophan residues,
with low level of tryptophan, it slows down the reaction so that RNA polymerase stays
on the DNA and also code the trpEDCBA, but at high levels, RNA pol. falls of DNA
template. Because the translation and transcription are happening at the same time in
prokaryotes.
o Trp-attenuator structure can form two different conformations. One of the
conformations forms a terminator loop, which ends the transcription. It depends on the
ribosome and the tryptophan level. When tryptophan is low, ribosome stalls at trp
codons and transcription is completed but if tryptophan level is high, ribosome
finishes leader peptide and for termination loop.
Gene Activator Repressor
lac CAP (cooperative) Lac repressor (cooperative)
gal CAP galR (cooperative)
glnA NtrC (allosteric)
merT MerR + Hg (DNA twist) MerR w/out Hg
 (allosteric)
PA3 prom. and polym. P4
PA3 and PA2C prom.s P4
araBAD prom. AraC and CAP

THE CASE OF BACTERIOPHAGE λ:

● Phage dormant prophage can switch lysogenic to lytic growth effectively under

DNA damage. Phenomena called lysogenic induction.

● Lytic when PL and PR promoters on, PRM (promoter for repressor maintanence)

off. Lysogenic when its vice versa.

● cI gene encodes λ repressor, which is dimer. NTD binds DNA, CTDs bind dimers

forming tetramer. Those dimers bind operators below cooperatively as well as

each other.

● λ repressor can activate like CAP or repress like lac repressor.

● Cro is just repressor. Has single domain and monomers form dimer to function.

● 6 operaters total, 3right and 3left hand control region.

● Or1, Or2 and Or3 are operators of right one.

● Repressor binds Or1 tenfold better than others.

● Cro binds Or3 likewise.

● In lysogenic growth, repressor bind Or2 and Or1. The one on Or2 contacts RNA

pol. at PRM, leading cI gene expression. Also turning off PR an PL.

● In lytic, Cro bind Or3 on the PRM, repress it and express cro gene from PR. If

neither repressor nor Cro bound, PR and PL (strong promotors) direct lytic growth.

● Lysogenic induction
 o RecA stimulates autocleavage of CTD of repressor.
o (RecA cleaves also LexA repressor, which represses the DNA-repair
genes.)
o It cannot form tetramer and loses cooperativity. Going of from Or2 and
Or1.
o The loss of repression leads transcription from PR and PL.
o PR produces cro and the lytic growth becomes permanent.

● Keeping its own transcription high is positive autoregulation. Low is negative

autoregulation.
o PRM activated by repressor on Or2 leading its own transcription. But if it
is too high amount, it also binds Or3 and repress PRM.
o Also repressors of Or and Ol dimers may bind and form octomers though
looping the DNA. This increases the repression of PL and PR, stabilising
lysogenic state. On the other hand, the repressor could bind Or3 at a lower
concentration than it would normaly does (long distance negative
autoregulation).
o The small decreases of repressor amount compansated by increased
expression. While small increases by switching the gene off.

● The initial decision whether to go lytic or lysogenic after infecting host resulated

by c2 and c3 genes on phage.

● c2 transcribed from PR, c3 from PL. C2 protein is activator and binds the upstream

promoter PRE (for repressor establishment) and stimulates transcription of c1 gene

(repressor).

● PRE is a weak promoter because it has very poor -35. The C2 protein binds there

but in opposite faace of the DNA helix, directly interacts with RNA pol. and helps
binding to promoter.

● Only if it produces enough reprossor, the repressor go bind Or1 and Or2 and

induce its own transcription.

● After infected initially;

o PR and PL constitutive promoters work immediately.

 o PR produce Cro and C2.

▪ After Cro produced high enough, it binds Or3 and block PRM.

Lytic development.

▪ C2 on the other hand transcribe repressor and to achieve successful

lysogenic state, repressor binds Or1 and Or2, activate PRM.

● If the phage number is high, it favors lysogenic development.

● C2 is very unstable because it is degraded by protease called FtsH (Hflb) encoded

by hfl gene. Thus reprosser synthesis is high if the FtsH is low. If that gene is
absent, almost always lysogenic. FtsH activity regulated by growth conditions.
o If growth is good, FtsH very active, C2 degraded a lot, repressor can not
be made, phage grow lytically.

● C2 regulation also regulated by C3 protein. It stabilises C2 because it is

alternative substrate for FtsH.

● Another C2-dependent promoter, PI located in front of gene int, encodes integrase

enzyme that catalyses formation of prophage.

● PAQ located middle of the gene Q, retards lytic growth, promotes lysogenic

growth.
o PAQ RNA acts as antisense message to Q message and promotes its
degredation. Q protein promotes late stage of lytic growth.

● Antitermination

o Is positive transcriptional regulation.

o N protein act on 3 terminators:

▪ One to the left of the N gen itself, one to the right of cro, one

between genes P and Q.

▪ It prevents termination only in the early operons of λ but not in

other bacterial or phage operons.

 ▪ Recogniton sequence is between promoter and th terminator, called

nut sites (N utilization).

▪ N binds to RNA transcribes from DNA that contains nut sequence.

Then it loads in the polymerase and prevent termination.

▪ NusA, NusE, and NusG proteins form complex on nut sequence

working together with N. However N can work itself just fine.

o Q protein act on only one terminator, 200 n downstream from the late
gene promoter PR’.

▪ Q protein recognise DNA sequences (QBE) between -10 and -35

regions of the late gene promoter (PR’).

▪ If Q not present, after initial 16-17n , the polymerase continues but

terminates at terminator (tR’).

▪ If Q present, it binds to QBE sequence and joins the polymerase.

With Q, the pol can transcribe through tR’.

● Retroregulation

o C2 protein activates PI promoter that directs int gene expression. It express

enzyme to integrate phage genome into host genome for lysogenic state
(integrase).
o int gene transcribed from both PI and PL but later one is degraded while
the former one is stable.

▪ PI transcript stops after terminator 300 n after the end of int gene.

Form stem-and-loop followed by 6 uridines.

▪ PL transcript goes under N protein protection and transcripts goes

way beyond terminator. The stem is forms would be substrate for

nucleases. Because the degredation proceeds backwards through
gene, it is called retroregulation.
o When a prophage is induced, integrase and excisionase must be produced
to catalyse reformation of free phage DNA by recombination out of the
bacterial DNA. However it would be in low C2 level.

▪ When the phage intagrates itself to bacterial genome, the

attachment site would be between int gene and stem site that leads
 degredation. Thus in the intergrated form, PL can produce stable int
mRNA.

Protein Activated Promoters (or Favored growth

function )
Repressor PRM lysogenic
Cro PR and PL lytic
RecA PR and PL lytic
C2 PRE (then PRM) lysogenic
FtsH Degrades C2 lytic
C3 Substrate instead C2 lysogenic