JIB 322

11 February 2017

Gene regulation in
Prokaryotes

Dr Mustafa Fadzil Farid Wajidi

Pusat Pengajian Pendidikan Jarak Jauh
• Some genes are constitutive i.e. expressed at
all times but others are regulated/ inducible
• Gene regulation important so that gene(s)
produce proteins that they code for at specific
times/ situations.
• Gene regulation provides economy of
resources for cell/ organism i.e. produce
according to demand
• In general, regulated genes may be involved in
i) metabolism; ii) response to environmental
stress; iii) cell division; iv) response to changes
in physiology e.g. cancer
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 Transcriptional regulation
• Transcriptional regulation involves the actions of
regulatory proteins that bind to the DNA and
affect the rate of transcription.
• a. Repressors inhibit transcription @ negative
control.
• b. Activators increase transcription @
positive control.
• Effector molecules do not interact directly with
the DNA, but rather directly with the repressors
or activators. This usually results in a
conformational change in the activator or
repressor.

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• Effector molecules are named by how they affect
transcription when they are present in the cell.
Regulatory proteins are named by how they interact
with the DNA.
• a. Inducers are effector molecules that cause
an increase in transcription. Genes that are regulated
by inducers are called inducible.
• b. Corepressors are effector molecules that
bind to repressors, causing the repressor to bind to the
DNA. They decrease the rate of transcription.
• c. Inhibitors bind to an activator protein and
prevent it from binding to the DNA. Genes that are
regulated in this manner are called repressible.

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 Inducer causes
repressor to
release DNA

Inducer causes
activator to
bind to DNA

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 Corepressor
causes repressor
to bind to DNA

Inhibitor causes
activator to
detach from DNA

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• Enzyme adaptation means that an enzyme
appears in the cell only after the cell has been
exposed to the substrate for that enzyme.
• Jacob and Monad examined lactose metabolism
in E. coli. Their experiments indicated that:
• Exposure to lactose causes a 1,000 to
10,0000-fold increase in the synthesis of lactose
metabolizing enzymes.
• The removal of lactose abruptly terminated
the synthesis of those enzymes.
• By studying mutations they concluded that a
separate gene encoded for each enzyme.

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 The lac Operon
• In bacteria, structural genes are often arranged
together under the control of a single promoter.
This is called an operon.
• Transcription of an operon creates a polycistronic
mRNA, which contains instructions for two or
more structural genes.
• Operons have a promoter at the beginning and a
terminator at the end.
• In addition, there is an operator site that controls
the ability of the RNA polymerase to transcribe
the operon.

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• The lac operon contains the following
components:
• lacP – the promoter
• lacO – the operator
• lacZ – encodes β-galactosidase, which
enzymatically cleaves lactose.
• lacY – encodes a lactose permease, which
actively transports lactose into the cell.
• lacA – encodes a transacetylase enzyme
that modifies the lactose
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• Two regulatory sites are located near the lac
operon.
• The operator provides a location for repressor
proteins to bind
• The CAP site is a DNA sequence recognized by
an activator protein called catabolite activator
protein (CAP).
• The lacI gene (not part of the operon)
encodes a lac repressor, which is transcribed
at low levels in the cell.

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 negative control
• The lac operon is regulated in several
manners.
• The lac repressor protein binds to the
operator and prevents the RNA
polymerase from recognizing the
promoter and transcribing the operon.
• This is an example of an inducible,
negative control mechanism.

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 Induction of lac operon

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• The binding of allolactose produces a
conformational change in the lac repressor,
preventing it from binding to the operator and
allowing transcription.
• Binding to a site on the enzymatic apart from
the active site and inducing a conformational
change in the enzyme is known as allosteric
regulation.

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 Induction of lac operon

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• Figure illustrates the progression of events from
the absence of lactose to the time when lactose
is introduced into the cell.
• The lac repressor has a measurable affinity for
allolactose. The likelihood that allolactose will
bind to the repressor is dependent on the
concentration of the allolactose.
• During induction of the operon, the
concentration of the allolactose rises, making it
more likely that the allolactose will bind to the
repressor, causing it to release from the operator.
• As the cell utilizes lactose, the allolactose levels
will fall, allowing the repressor to bind again to
the operator.

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• Jacob, Monad and Pardee determined that
the lacI gene encodes a repressor protein, and
is not part of the operon.
• Their experimental system tested for one of
two possibilities:
• The lacI gene encodes a repressor protein.
• The lacI gene acts as a binding site for a
repressor protein.

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• Jacob et al. used bacteria that contained small
circular pieces of DNA called F factors.
• These F factors sometimes carry genes from the
bacterial chromosome. This type of F factor is
called a F’ factor and the cells that contain them
are called merozygotes.
• By utilizing mutations, normal and merozygote
lines, the researchers were able to distinguish
between the two hypothesis.
• Their experiment indicated that the lacI gene
does not need to be located near the operon to
influence activity, and thus is not the binding site
for a repressor protein. Rather, it encodes a
repressor protein that diffuses to the operon.
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 cis & trans factors
• A trans-acting factor does not require the
DNA (gene) producing the factor to be in
close proximity to its site of action. Proteins
are able to work “in trans”.
• A cis-effect occurs when the DNA segments
(genes) must be physically connected. DNA
sequences are cis-acting factors.

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 Activator Proteins and the lac Operon
• lac operon is also regulated by catabolite repression. The
presence of glucose in the cell represses the lac operon.
Glucose is a catabolite of cellular metabolism.
• If placed in an environment containing both glucose and
lactose, E.coli will use the glucose first (diauxic growth).
• The effector molecule for this pathway is cyclic AMP (cAMP).
This molecule is produced from ATP by adenyl cyclase.
• cAMP binds to an activator protein called CAP (catabolite
activator protein).
• This is an inducible system, under positive control.
• The binding of cAMP to CAP causes the cAMP-CAP complex to
bind to the CAP site near the lac promoter and increases the
rate of transcription.

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 The ara Operon
• The ara operon in E. coli is involved with
arabinose (sugar).
• The AraC protein can act as a repressor or
activator of transcription, depending on the
presence of arabinose.
• In the absence of arabinose, AraC acts as a
repressor.
• In the presence of arabinose, AraC acts as
an activator.

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 ara operon

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 The trp Operon
• The trp operon encodes enzymes that are needed for
the biosynthesis of the amino acid tryptophan.
• trpL and trpR are involved in regulation.
• trpE, trpD, trpC, trpB and trpA encode enzymes
for biosynthesis.
• trpR encodes the trp repressor.
• When tryptophan levels are low, the trp repressor does
not bind to the operator site, and transcription of the
operon proceeds.
• When tryptophan is present, it acts as a corepressor,
binding to the trp repressor and allowing it to bind to
the operator. This inhibits transcription.

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 Regulation via the trp repressor

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 Attenuation
• Attenuation, was discovered by Yanofsky (1970s)
by studying mutant strains that lacked the trp
repressor.
• Attenuation occurs in bacteria due to the
coupling of transcription and translation.
• During attenuation, transcription begins, but
is not finished.
• An area of DNA called the attenuator
regulates this process and stops transcription of
the tryptophan biosynthesis enzymes.
• Several metabolic pathways for amino acid
production are regulation through attenuation.

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Translational and Posttranslational Regulation
• The majority of gene regulation in bacteria is
at the transcriptional level.
• Some regulation occurs during initiation,
elongation, and termination of translation.
• Posttranslational regulation refers to the
functional control of proteins that are already
present in the cell.

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 Repressor Proteins and Antisense RNA
• The translation of mRNA can be influenced by
proteins that influence the ability of the
ribosome to form a polypeptide.
• Translational regulatory proteins recognize
sequences within the mRNA. These are called
translational repressors.
• Translational repressors may inhibit
translation by binding to the Shine-Dalgarno
sequence, or by binding to a region of the
mRNA that promotes an RNA secondary
structure that inhibits translation.
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 Regulation by antisense
• Antisense RNA is a piece of RNA that is
complementary to the mRNA.
• An example is osmoregulation in E. coli in
which the production of antisense RNA
inhibits translation of the mRNA.

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 Posttranslational Regulation
• Feedback inhibition is another mechanism of
regulation for metabolic enzymes.
• The final product of the pathway inhibits the
activity of one or more enzymes in the pathway.
• An allosteric enzyme has two different binding
sites. The catalytic site is responsible for the
binding of the substrate. The regulatory site
allows for a means of turning the enzyme off,
usually by a conformational change to the
enzyme and catalytic site.
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• Enzymes may also be modified by
covalent modification of their structure.
• This may include proteolytic processing,
disulfide bond formation, or the
attachment of sugars, functional groups
or lipids to the enzyme.
• This process is known as
posttranslational covalent modification.

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 Gene Regulation in Phage λ
• Phage λ may infect bacteria either by the
lytic or lysogenic cycles.
• In the lysogenic cycle, the phage acts as a
temperate phage, and integrates its
genetic material into the bacterial
chromosome, forming a prophage.
• The genome of phage λ is linear until it is
injected into the host bacteria.
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 Lysogenic Cycle of Phage λ
• Regulatory proteins determine the
choice between the lytic and lysogenic
cycles.
• After infection, two promoters, PL and PR,
are activated which begins a competition
between the lytic and lysogenic cycles.

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• Transcription produces a cro protein and the N
protein.
• N protein has an antitermination function. It binds
to the RNA polymerase and prevents termination.
• The transcript contains the PR promoter and
includes several additional genes, viz, activator
protein (cII), genes for initializing phage synthesis
(O and P) and a second antiterminator (Q).
• The N protein also interacts with the PL promoter,
extending the transcript to include genes for
integrating the phage genome into the bacterial
chromosome (int), an excise gene (xis) and a
stabilizer for the cII protein (cIII).
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 Lysogenic Cycle of Phage λ
• If the cII-cIII complex accumulates to sufficient
levels, then the lysogenic cycle is favoured.
• The cII protein activates the a gene that
encodes the λ repressor (cI). It also activates
the int gene. These two proteins promote the
lysogenic life cycle.
• The λ repressor inhibits expression of cI since
the levels of λ repressor are quickly elevated,
which further inhibits the lytic cycle.

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 Lytic Cycle of Phage λ
• In order for the lytic cycle to commence, the λ
repressor must be prevented from inhibiting
the lytic cycle. This is the function of the cro
protein.
• The accumulation of cro protein prevents the
expression of the cI gene, which encodes for
the λ repressor.
• The cro protein also allows for the
transcription of the O, P, and Q genes, which
are involved in the replication of λ DNA.
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• The determination of the lytic or lysogenic cycles
is made early.
• cII protein is easily degraded by cellular proteases
produced by E. coli. These proteases are
abundant if the growth conditions are favorable
for the bacteria.
• If cII is degraded, λ repressor is not made and
the lytic cycle is favored.
• Starvation conditions (poor nutrients) favours the
lysogenic cycle, since cII will build up faster,
promoting the formation of the λ repressor.
• The lytic cycle may be induced by UV exposure.
• UV exposure increases transcription of recA.
• recA inactivates λ repressor by cleavage.
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 The Genetic Switch of OR
• The OR region contains three operators.
• cro and λ repressor can bind to all three
sites.
• These proteins determine the switch
between the lytic and lysogenic cycles.
• During the lysogenic cycle, the λ repressor
controls the switch, but during the lytic cycle
the cro protein controls the switch.

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