Transcript The lac repressor binds to operator DNA

PowerPoint to accompany
Genetics: From Genes to
Genomes
Fourth Edition
Leland H. Hartwell, Leroy Hood,
Michael L. Goldberg, Ann E. Reynolds,
and Lee M. Silver
Prepared by Mary A. Bedell
University of Georgia
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PART V
How Genes Are Regulated
CHAPTER
Gene Regulation in
Prokaryotes
CHAPTER OUTLINE




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15.1 Overview of Prokaryotic Gene Regulation
15.2 The Regulation of Gene Transcription
15.3 Attenuation of Gene Expression: Termination of Transcription
15.4 Global Regulatory Mechanisms
15.5 A Comprehensive Example: The Regulation of Virulence Genes in V. cholerae
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RNA polymerase participates in all three
phases of transcription
Initiation – core RNA polymerase plus sigma (σ) factor
• Core has four subunits: two alpha (α), one beta (β), one beta
prime (β')
• DNA is unwound and polymerization begins
Elongation – core RNA polymerase without σ factor
• Continues until RNA polymerase recognizes termination
signal
Termination – two kinds in bacteria
• Rho-dependent – Rho (ρ) protein binds to RNA polymerase
and removes it from RNA
• Rho-independent – 20 nt sequence in RNA forms stem-loop
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Role of RNA polymerase in initiation and
elongation phases of transcription
Fig. 15.2
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Two kinds of transcription termination
in bacteria
Fig. 15.2 (cont)
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Regulation of expression can
occur at many steps
Transcriptional control
• Binding of RNA polymerase to promoter
 Most critical step in regulation of most prokaryotic genes
• Shift from initiation to elongation
• Release of mRNA at termination
Posttranscriptional control
• Stability of mRNA
• Efficiency of translation initiation
• Stability of polypeptide
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Utilization of lactose by E. coli provides
a model system of gene regulation
Lactose utilization requires two enzymes (Fig. 15.3)
• Permease transports lactose into cell
• β-Galactosidase (β-Gal) splits lactose into glucose and
galactose
In the absence of lactose, both enzymes are present at very
low levels
Lactose is the inducer of the genes encoding permease
and β-Gal
• Induction – stimulation of synthesis of a specific protein
• Inducer – molecule responsible for induction
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Lactose utilization in an E. coli cell
Fig. 15.3
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Advantages of using lactose utilization by E. coli
as a model for understanding gene regulation
Lac− mutants can be maintained on media with glucose and
so lac genes are not essential for survival
• If both glucose and lactose are present, E. coli cells will use
glucose first
Simple assays for lac expression - use of ONPG or X-gal as
substrates for β-gal (color change)
Lactose induces a 1000-fold increase in β-gal activity
Detection and characterization of hundreds of lac− mutants
defective in lactose utilization
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Studies of lac− mutants revealed
the operon theory of gene regulation
Jacques Monod and Francois Jacob – Pasteur Institute
• Nobel Prize in 1965 (with A. Lwoff) for their discoveries
concerning genetic control of enzyme and virus synthesis
• Compared the effects of many different types of lac mutants
on induction and repression of enzyme activity for lactose
utilization
• Operon theory - one signal can simultaneously regulate
expression of several clustered genes
• Hypothesized that lac genes are transcribed together as a
single mRNA (polycistronic) from a single promoter
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The lactose operon in E. coli
The players
• Three structural genes - lacZ, lacY, and lacA
• Promoter - site to which RNA polymerase binds
• Cis-acting operator site – controls transcription initiation
• Trans-acting repressor - binds to the operator (encoded by
lacI gene)
• Inducer - prevents repressor from binding to operator
Fig. 15.5a
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Repression of lac gene expression
In the absence of lactose, repressor binds to the operator
and prevents transcription
lac repressor is a negative regulatory element
Fig. 15.2b
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Induction of the lac
operon in E. coli
When lactose (or IPTG) is
present:
• Inducer binds to the lac
repressor
• Repressor changes
shape and cannot bind
to operator
• RNA polymerase binds
to the promoter and
initiates transcription of
the polycistronic lac
mRNA
Fig. 15.2c
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Jacob and Monod defined the roles of the lac
genes by genetic analysis of many lacI− mutants
Complementation analysis identified three genes in a tightly
linked cluster
• lacZ encodes β-galactosidase
• lacY encodes permease
• lacA encodes transacetylase
• Most studies focused on lacZ and lacY
Constitutive expression of β-galactosidase and permease
was caused by mutations in the lacI gene
• Constitutive mutants (lacI−) express the enzymes in the
absence and presence of inducer
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The PaJaMo experiment provided evidence
that lacI encodes a repressor
lacI+ lacZ+ DNA transferred
into lacI− lacZ− cells
β-gal levels increased initially
β-gal levels decreased as
repressor accumulated
β-gal accumulation resumed
after addition of inducer
Fig. 15.7
Jacob and Monod proposed that lacI encodes a repressor
that binds to an operator site near the lac promoter
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How the inducer acts to trigger synthesis
of lac enzymes
Binding of inducer to repressor changes the shape of the
repressor so that it can longer bind to DNA
• When no inducer is present, repressor is able to bind
to DNA
Repressor is an allosteric protein – undergoes reversible
changes in conformation when bound to another molecule
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lacl− mutants have a mutant repressor that
cannot bind to operator
In lacI− mutants, lac genes are expressed in the absence
and the presence of inducer (constitutive expression)
Fig. 15.8
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lacls mutants have a superrepressor that binds
to operator but cannot bind to the inducer
In lacIS mutants, lac genes are repressed in the absence and
the presence of inducer
Fig. 15.9
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lac repressor has two separate domains
Mutated sequences in
many different lacI−
mutants clustered in the
DNA-binding domain
Mutated sequences in
many different lacIS
mutants clustered in the
inducer-binding domain
X-ray crystallography
revealed the two
separate domains
Fig. 15.10
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lacOc mutants have a mutant operator that
cannot bind the repressor
In lacOc mutants, lac genes are expressed in the absence
and the presence of inducer (constitutive expression)
Fig. 15.11
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Proteins act in trans, DNA sites act in cis
Jacob and Monod used partial diploids carrying different
alleles of lac regulatory elements and structural genes to
identify trans-acting and cis-acting elements
• F' lac plasmids (Chapter 14) were used to generate
partial diploids
Trans-acting elements:
• Can diffuse through the cytoplasm and act at target
DNA sites on any DNA molecule in the cell
Cis-acting elements:
• Can only influence expression of adjacent genes on
the same DNA molecule
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Lacl+ protein acts in trans
Fig. 15.12
Repressor expressed from the plasmid can diffuse through the
cytoplasm and bind to the operator on the chromosome
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Lacls protein acts in trans
Fig. 15.13
Superrepressor expressed from the plasmid can diffuse through
the cytoplasm and bind to the operator on the chromosome, even
in the presence of inducer
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lacOc acts in cis
Fig. 15.14
The lacOC mutation affects expression of genes only on the DNA
that it is located on
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The lac operon of E. coli is regulated
by both lactose and glucose
When both glucose and lactose are present, only glucose is
utilized
Lactose induces lac mRNA expression, but only in the
absence of glucose
• Lactose prevents repressor from binding to lacO
• lac repressor is a negative regulator of lac transcription
lac mRNA expression cannot be induced if glucose is
present
• Glucose controls the levels of cAMP
• cAMP binds to cAMP receptor protein (CRP)
• CRP-cAMP is a positive regulator of lac transcription
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Positive regulation by CRP–cAMP
Catabolite repression – overall effect of glucose is to
prevent lac gene expression
Fig. 15.15
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Positive regulation of the araBAD
operon by AraC
Three structural genes required in the breakdown of the
sugar arabinose - araB, araA, and araD
• Arabinose genes are in an operon and are induced
when arabinose is present
AraC is a positive regulator of the araBAD operon
• Loss of function of AraC results in no expression of the
araBAD operon when arabinose was present
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AraC is a positive regulator
Fig. 15.16
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Further studies revealed more about
regulatory proteins and sites
Biochemical evidence for lac repressor binding to lacO (Fig. 15.17)
X-ray crystallography revealed the structure of repressor proteins
• lac repressor has a helix-turn-helix (HTH) motif (Fig. 15.18)
Evidence that specific amino acids in the α-helices of lac
repressor are required for binding to lacO (Fig. 15.19)
DNA sequences to which negative and positive regulators bind
have a two-fold rotational symmetry
• e.g. CRP-binding site of the lac operon
Most DNA-binding regulatory proteins are oligomeric, with two
to four subunits
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The lac repressor binds to operator DNA
Fig. 15.17
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DNA recognition sequences by
helix-turn-helix (HTH) motif
A protein with an HTH motif has
two α-helical regions separated
by a turn in the protein
The HTH motif fits into the
major groove of DNA
One of the α-helices recognizes
a specific DNA sequence
Fig. 15.18
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Changing amino acids in recognition sequence
of a repressor protein
434 repressor binds to an operator in the DNA of the 434 virus
P22 repressor binds to an operator in the DNA of the P22 virus
Amino acid sequences in the α-helix of 434 repressor were
modified to have amino acid sequence like that of P22 repressor
Hybrid 434-P22 functioned just like the P22 repressor
Fig. 15.19
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DNase footprint shows where proteins bind
Incubate radiolabeled DNA
from lac+ operon with
partially purified protein
from lacI+ cells
Partial digest of DNA with
DNase I
Gel electrophoresis and
autoradiography
If protein is bound to DNA,
then specific fragments will
be protected from DNase I
digestion
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Fig. 15.20
33
CRP–cAMP binds as a dimer to
a regulatory region
CRP-binding sites have a two-fold rotational symmetry
5'TGTGAGTTAGCTCACA 3'
3'ACACTCAATCGAGTGT 5'
CRP protein binds as a dimer
• CRP-binding site consists of two recognition
sequences, one for each subunit of the CRP dimer
Fig. 15.21
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lac repressor tetramer binds to two sites
lac repressor is a tetramer, with each subunit containing a
DNA-binding HTH motif
lac operon has three operators (O1, O2, and O3) each of
which contains two recognition sequences for lac repressor
• O1 has the strongest binding affinity for lac repressor
• Maximal repression occurs when all four repressor subunits
are bound
Two repressor subunits bind to O1
Two repressor subunits bind to
either O2 or O3
Fig. 15.2
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AraC acts as both a repressor and an activator
AraC can bind to three sites (araO, araI1, and araI2) with
different affinities
(a) No arabinose present:
When AraC is bound to araO
and to araI1, looping of DNA
occurs and prevents
transcription
(b) Arabinose present:
Arabinose causes allosteric
change in AraC so that it cannot
bind to araO
AraC interacts with RNA
polymerase only when both araI1
and araI2 are occupied
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Fig. 15.23
36
Interaction of regulatory proteins with
RNA polymerase
Many negative regulators (e.g. lac repressor) prevent
transcription initiation by blocking the functional binding of
RNA polymerase (Fig. 15.24)
Many positive regulators (e.g. CRP-cAMP) establish contact
with RNA polymerase that enhances transcription initiation
(Fig. 15.25)
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Overlapping binding sites for RNA polymerase
and lac repressor
When lac repressor is bound to lac operator, functional
binding of RNA polymerase to the promoter is blocked
Fig. 15.24
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CRP-cAMP complex makes direct contact
with RNA polymerase
Without interaction with CRP-cAMP, RNA polymerase can
bind to the promoter but is less likely to unwind DNA and
initiate transcription
Fig. 15.25
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Using the lacZ gene as a reporter
of gene expression
Reporter gene – protein-encoding gene whose expression
in the cell is quantifiable by sensitive and reliable
techniques
Measuring gene expression
• Fuse coding region of lacZ to cis-acting regulatory regions
from other genes (Fig. 15.26)
Identifying sets of genes regulated by the same stimulus
• Create library of cells with promoter-less lacZ inserted by
transposition into random sites in the genome (Fig. 15.27)
Controlling gene expression
• Fuse the lac regulatory sequences to the coding region of a
foreign gene (Fig. 15.28)
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Hartwell et al., 4th edition, Chapter 15
lacZ fusion used to perform genetic studies of
the regulatory region of gene X
Conditions that regulate expression of the test regions from
gene X will alter the levels of β-galactosidase
Specific regulatory sites can be identified by constructing
and testing mutations in the test regions of gene X
Fig. 15.26
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Using lacZ to identify sets of genes regulated
by the same stimulus
Fig. 15.27
Transposition of
promoter-less lacZ
coding region
Library of clones containing
lacZ insertions at random sites
Screen library to identify all the
genes that express lacZ in
response to a signal
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Use of fusions to
overproduce a gene
product
Expression of gene X under control
of the lac regulatory system
Fig. 15.28a
Expression of human growth
hormone in E. coli controlled by
lac control region
Fig. 15.28b
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Regulation of the tryptophan (trp)
operon in E. coli
Structural genes for tryptophan (Trp) biosynthesis are
expressed only in the absence of Trp
Two mechanisms for trp operon regulation
• TrpR gene encodes the trp repressor that can bind to
the Trp operator (TrpO)
 When Trp is present, TrpR repressor binds to TrpO
 When Trp is absent, TrpR repressor cannot bind to TrpO
• Attenuation controls termination of transcription in the
trp leader (TrpL)
 When Trp is present, transcription terminates in TrpL
 When Trp is absent, transcription doesn't terminate in TrpL
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Tryptophan acts as a corepressor
Binding of tryptophan to TrpR repressor allows TrpR to bind
to TrpO and inhibit transcription of the five structural genes
In the absence of tryptophan, TrpR repressor cannot bind to
TrpO
Fig. 15.29a
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Evidence that TrpR repressor is not the only
regulator of the trp operon
Constitutive expression of Trp biosynthesis doesn't occur
in TrpR− mutants
If TrpR were the sole regulator, maximal expression of trp
genes would occur in the absence or presence of
tryptophan
Second regulatory mechanism is attenuation – control of
gene expression by premature termination of transcription
Table15.1
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Transcription from the trp promoter produces
two alternative mRNAs
Attenuation controls termination of transcription in the trp
leader (TrpL)
• Truncated mRNA - terminates in TrpL, only 140 bases
• Full-length mRNA - continues through TrpL and
encodes all five structural genes
Fig. 15.29b
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Alternate stem-loop structures in trpL RNA
Different regions of trpL have complementary base-pairing
• Formation of the 1-2 stem-loop allows formation of the
3-4 stem-loop
• Formation of the 2-3 stem-loop prevents formation of
the 3-4 stem-loop
• The 3-4 stem loop is a transcription terminator
Fig. 15.30a
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When tryptophan is present, transcription
terminates in trpL because of translation
The trpL mRNA is translated and includes two trp codons
Movement of ribosomes through trpL mRNA depends on
the availability of tRNATrp
 When Trp is present, tRNATrp is available and rapid
ribosome movement allows the formation of 3-4 stem-loop
Fig. 15.30b
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When tryptophan isn't present, transcription
doesn't terminate in trpL
Fig. 15.30c
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Global regulatory mechanisms
Dramatic shifts in environmental conditions can trigger
expression of sets of genes or operons
Regulon – a group of genes whose expression is controlled
by the same regulatory protein
Two examples in E. coli:
• CRP-cAMP controls several catabolic operons
• Expression of several genes induced by heat shock
 Highly conserved stress response
 Induced proteins include those that recognize and degrade
aberrant proteins and chaperones, which assist in
preventing protein aggregation
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Sigma factor (σ) recognition sequences
Normal, housekeeping sigma factor is σ70
• Active under normal physiological conditions, but is
inactivated by heat shock
rpoH genes encodes σ32, an alternative sigma factor
• Heat shock inducible genes have promoters that are
recognized by σ32
• σ32 is resistant to inactivation by heat shock
Fig. 15.31
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Factors influencing increase in σ32 activity
after heat-shock treatment
Increased transcription of rpoH gene
Increased translation of σ32 mRNA because of increased
stability of rpoH mRNA
Increased stability and activity of σ32 protein
No longer inhibited by chaperones DnaJ/K
No competition from σ70 because it is removed by
degradation
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Alternate sigma factor in the
heat-shock response
At normal temperatures, promoter for rpoH gene is
recognized by σ70
After heat shock, σ70 is degraded and transcription of the
gene for σ24 is increased
• σ24 recognizes a different promoter sequence at rpoH
• Increased expression of σ32 causes transcription of
several genes
Fig. 15.32
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Translational control of another sigma factor
encoded by the rpoS gene
Under normal conditions, rpoS gene is transcribed but rpoS
mRNA is not translated
After stress response, a small RNA (dsrA) binds to rpoS
mRNA and allows translation
Fig. 15.33
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Tools for studying genes regulated
in a global response
Microarrays of expression in different growth conditions,
e.g. E. coli grown on glucose, glycerol, succinate, or alanine
 Switch from glucose to glycerol or succinate caused
increased expression of 40 genes
 Switch from glucose to alanine caused increased
expression of 188 genes
Mutants in specific genes, e.g. NtrC is a master control gene
activated by lack of ammonia
Computer analysis to identify regulatory proteins, e.g.
searches for HTH DNA binding motif
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A comprehensive example: The regulation of
virulence genes in V. cholerae
V. cholerae is the bacterial species that causes cholera, a
life-threatening diarrheal disease
Bacteria are ingested in contaminated drinking water
Respond to changes in environment by increasing or
decreasing transcription and/or translation of specific
genes
In intestine, V. cholerae express proteins to make flagella
and to degrade mucous so that they can reach epithelial
cells
Once the bacteria reach the epithelial cells, they secrete
toxins that result in diarrhea
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Identification of regulators of toxin
production in V. cholerae
Two genes, ctxA and ctxB, identified that encode subunits
of cholera toxin
Gene fusions of ctxA promoter and lacZ coding region
created and transformed into E. coli
Transformation of E. coli with ctxA-lacZ reporter gene with
fragments of V. cholerae genomic DNA
toxR gene from V. cholerae identified in genomic DNA that
caused increased expression of ctxA-lacZ reporter gene
Further experiments showed that mutation of toxR gene in
V. cholerae abolished its virulence
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Identification of other V. cholerae genes
regulated by toxR
Library of V. cholerae cells created that had random
insertions of lacZ coding region
Gene fusions of a constitutive promoter and toxR coding
region created and transformed into the lacZ library
Colonies with high β-gal expression had lacZ sequences
inserted adjacent to promoters regulated by toxR
In E. coli, toxR could not affect expression of the same
genes
ToxT was then identified as a positive regulator of many V.
cholera virulence genes
TcpP and ToxR both bind to ToxT promoter and are both
required for ToxT transcription
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Model for how V. cholerae regulates
genes for virulence
Fig. 15.34
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Unanswered questions about expression of
virulence genes in V. cholera
What is the signal that makes cholera bacteria stop
swimming and start colonizing the intestinal epithelial
cells?
What molecular events differentiate swimming versus
adherence?
Why is there a cascade of regulatory factors (ToxR and
ToxT)?
A better understanding of V. cholerae pathogenesis will lead
to more effective treatments and preventatives for cholera
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