Transcript Document

Chapter 17
Regulation of Gene
Expression in Bacteria and
Bacteriophages
Copyright © 2010 Pearson Education Inc.
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Growth and division genes of bacteria are regulated genes. Their
expression is controlled by the needs of the cell as it responds to
its environment with the goal of increasing in mass and dividing.
Genes that generally are continuously expressed are constitutive
genes (housekeeping genes). Examples include protein synthesis
and glucose metabolism.
All genes are regulated at some level, so that as resources
dwindle the cell can respond with a different molecular strategy.
Prokaryotic genes are often organized into operons that are
cotranscribed. A regulatory protein binds an operator sequence
in the DNA adjacent to the gene array and controls production of
the polycistronic (polygenic) mRNA.
Gene regulation in bacteria and phage is similar in many ways to
the emerging information about gene regulation in eukaryotes,
including humans. Much remains to be discovered; even in E.
coli, one of the most closely studied organisms on earth, 20% of
the genomic ORFs have no attributed function.
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An inducible operon responds to an inducer
substance (e.g., lactose). An inducer is a small
molecule that joins with a regulatory protein to
control transcription of the operon.
The regulatory event typically occurs at a specific
DNA sequence (controlling site) near the proteincoding sequence.
Control of lactose metabolism in E. coli is an
example of an inducible operon.
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E. coli expresses genes for glucose metabolism constitutively, but the
genes for metabolizing other sugars are regulated in a “sugar specific”
way. Presence of the sugar stimulates synthesis of the proteins needed.
Lactose is a disaccharide (glucose + galactose). If lactose is E. coli’s sole
carbon source, three genes are expressed:
◦ a. b-galactosidase has two functions:
 i. Breaking lactose into glucose and galactose. Galactose is converted to glucose, and
glucose is metabolized by constitutively produced enzymes.
 ii. Converting lactose to allolactose (an isomerization). Allolactose is involved in
regulation of the lac operon.
◦ b. Lactose permease (M protein) is required for transport of lactose across the
cytoplasmic membrane.
◦ c. b-Galactoside transacetylase transfers an acetyl group from acetyl-CoA to bgalactoside for reasons that are not understood.
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The lac operon shows coordinate induction:
◦ a. In glucose medium, E. coli normally has very low
levels of the lac gene products.
◦ b.When lactose is the sole carbon source, levels of
the three enzymes increase coordinately
(simultaneously) about a thousandfold.
 i. Allolactose is the inducer molecule.
 Ii. The mRNA for the enzymes has a short half-life.
When lactose is gone, lac transcription stops, and
enzyme levels drop rapidly.
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Lac genes.
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a. b-galactosidase is lacZ.
b. Permease is lacY.
c. Transacetylase is lacA.
d. The genes are tightly linked in the order: lacZ-lacY-lacA.
The three genes are transcribed on one polycistronic
(polygenic) RNA. Premature translation termination
prevents this by reducing translation of the
downstream genes
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The operon regulation
◦ lac operator (lacO) just upstream from the lacZ gene.
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Upstream of lacO is the lac repressor gene (lacI).
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lacI gene is constitutively expressed at low levels
(weak promoter)
Without lactose lac repressor proteins bind to the
lacO (operator) NEGATIVE REGULATION.
No lactose- no induction
However, leaky lac operator produces few
molecules of the lacZ, lacY and lacA.
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b-galactosidase in wild-type E. coli growing with
lactose as the sole carbon source converts lactose into
allolactose.
◦ i. Repressor bound with allolactose changes shape (allosteric
shift) and dissociates from the lac operator. Free repressor–
allolactose complexes are unable to bind the operator.
◦ ii. Allolactose induces expression of the lac operon by removing
the repressor and allowing transcription to occur.
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The lacOC mutations
result in constitutive
gene expression.
They are cisdominant to lacO+,
because repressor
cannot bind to the
lacOC operator
sequence
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The lacI- mutations change the
repressor protein’s conformation and
prevent it from binding the operator,
resulting in constitutive expression of
the operon.
◦ a. In a partial diploid (lacI+ lacO + lacZ- lacY +
/lacI- lacO + lacZ + lacY-), the wild-type
repressor (lacI+) is dominant over lacImutants.
◦ b. Defective lacI- repressor can’t bind either
operator, but normal repressor from lacI +
binds both operators and regulates
transcription, resulting in functional bgalactosidase and permease
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a. Binding to the operator region
b. Binding with the inducer (allolactose)
c. Binding of repressor polypeptide subunits to
form an active tetramer
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Repressor exerts negative
control by preventing
transcription. Positive control
of this operon also occurs
when lactose is E. coli’s sole
carbon source, with no
glucose present.
◦ a. Catabolite activator protein
(CAP) binds cyclic AMP (cAMP).
◦ b. CAP–cAMP complex is a
positive regulator of the lac
operon. It binds the CAP site, a
DNA sequence upstream of the
operon’s promoter.
◦ c. Binding of CAP–cAMP complex
recruits RNA polymerase to the
promoter, leading to
transcription.
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When both glucose and lactose are in the
medium, E. coli preferentially uses glucose, due
to catabolite repression.
◦ a. Glucose metabolism greatly reduces cAMP levels in the
cell.
◦ b. The CAP–cAMP level drops and is insufficient to
maintain high transcription of the lac genes.
◦ c. Even when allolactose has removed the repressor
protein from the operator, lac gene transcription is at
very low levels without CAP–cAMP complex bound to the
CAP site.
◦ d. Experimental evidence supports this model. Adding
cAMP to cells restored transcription of the lac operon,
even when glucose was present.
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If amino acids are available in the medium, E.
coli will import them rather than make them, and
the genes for amino acid biosynthesis are
repressed. When amino acids are absent, the
genes are expressed and biosynthesis occurs.
Unlike the inducible lac operon, the trp operon is
repressible. Generally, anabolic pathways are
repressed when the end product is available.
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a. There are five structural genes, trpA through trpE.
b. The promoter and operator are upstream from the trpE
gene.
c. Between trpE and the promoter-operator is trpL, the leader
region. Within trpL is the attenuator region (att).
d. The trp operon spans about 7 kb. The operon produces a
polygenic transcript with five structural genes for tryptophan
biosynthesis.
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Two mechanisms regulate expression of the trp
operon:
◦ a. Repressor–operator interaction.
◦ b.Transcription termination.
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When tryptophan is present, it will bind to an
aporepressor protein (the trpR gene product).
◦ a. The active repressor (aporepressor plus tryptophan)
binds the trp operator, and prevents transcription
initiation.
◦ b.Repression reduces transcription of the trp operon
about 70-fold.
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When tryptophan is limited, transcription is also
controlled by attenuation.
◦ a. Attenuation produces only short (140 bp) transcripts
that do not encode structural proteins.
◦ b.Termination occurs at the attenuator site within the
trpL region.
◦ c. The proportion of attenuated transcripts to fulllength ones is related to tryptophan levels, with more
attenuated transcripts as the tryptophan concentration
increases.
◦ d.Attenuation can reduce trp operon transcription 8to 10-fold. Together, repression and attenuation
regulate trp gene expression over a 560- to 700-fold
range.
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The molecular model for attenuation:
◦ a. Translation of the trpL gene produces a short polypeptide.
Near the stop codon are two tryptophan codons.
◦ b. Within the leader mRNA are four regions that can form
secondary structures by complementary base-pairing
 i. Pairing of sequences 1 and 2 creates a transcription pause signal.
 Ii. Pairing of sequences 3 and 4 is a transcription termination signal
(a rho-independent terminator).
 Iii. Pairing of 2 and 3 is an antitermination signal, and so
transcription will continue.
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Tight coupling of transcription and translation in prokaryotes
makes control by attenuation possible.
◦ i. RNA polymerase pauses when regions 1 and 2 base-pair just
after they are synthesized
◦ ii. During the pause, a ribosome loads onto the mRNA and begins
translation of the leader peptide. Ribosome position is key to
attenuation:
◦ (1) When tryptophan (Trp) is scarce:
 (a) Trp–tRNAs are unavailable, and the ribosome stalls at the Trp codons
in the leader sequence, covering attenuator region 1.
 (b) When the ribosome is stalled in attenuator region 1, it cannot basepair with region 2. Instead, region 2 pairs with region 3 when it is
synthesized.
 (c) If region 3 is paired with region 2, it is unable to pair with region 4
when it is synthesized. Without the region 3–4 terminator, transcription
continues through the structural genes.
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(2) When Trp is abundant:
◦ (a) The ribosome continues translating the leader peptide,
ending in region 2. This prevents region 2 from pairing
with region 3, leaving 3 available to pair with region 4.
◦ (b) Pairing of regions 3 and 4 creates a rho-independent
terminator known at the attenuator. Transcription ends
before the structural genes are reached.