Transcript Binds to, DNA

Gene Regulation in
Prokaryotes
Outline of Chapter 16
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There are many steps in gene expression and regulation
can occur at any one of them
Genetic and molecular studies show that most regulation
affects the initiation of RNA transcripts

Studies of genes for lactose utilization
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Negative regulation – blocks transcription
Positive regulation – increases transcription
DNA binding proteins acting on RNA polymerase at promoter are main
agents of regulation
Attenuation of expression – tryptophan pathway

Gene expression is fine tuned by premature termination of
transcription
Outline of Chapter 16

Global regulatory mechanisms: E. coli’s response
to heat shock is an example of the bacterial
ability to coordinate the expression of different
sets of genes dispersed around the chromosome.
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Microarray analysis is an important new tool for
detecting changes in gene expression in response to
environmental changes
Comprehensive example: How Vibrio cholerae
regulate their virulence genes
RNA polymerase is the key enzyme
for transcription

RNA polymerase involved in three phases of
transcription

Initiation – sigma subunit + core enzyme (two alpha, one beta,
and one beta’ subunit)
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Binds to promoter, unwinds DNA, begins polymerization of bases
complementary to DNA template
Elongation – movement away from promoter sigma subunit
released, polymerization
Termination –signal reached by RNA polymerase
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Rho dependent termination – Rho factor recognizes sequence in mRNA,
binds to it, and pulls it away from RNA polymerase
Rho independent termination – stem loop structure formed by sequence
of 20 bases with a run of 6 or more U’s signals release of RNA
polymerase
Fig. 16.2

Translation in prokaryotes starts before transcription
ends
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Initiation sites for translation signal ribosomes to bind near 5’
end of mRNA while downstream transcription is still occurring
Polycistronic mRNAs often lead to the translation of several
genes at the same time from one mRNA transcript
The regulation of gene expression can occur at many
steps
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Binding of RNA polymerase to promoter
Shift from initiation to elongation
Release of mRNA at termination
Posttranscriptional stability of mRNA
Efficiency of ribosomes to recognize translation initiation sites
Stability of polypeptide product
The utilization of lactose by E. coli:
A model system for gene regulation

The presence of lactose induces expression of the genes
required for lactose utilization

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Induction – stimulation of protein synthesis
Inducer – molecule that stimulates synthesis
Lactose – inducer of genes for lactose utilization
1950s and 1960s – Golden era of bacterial genetics

Advantages of E. coli and lactose utilization system

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Culture large numbers of bacteria allow isolation of rare mutants
Lactose genes not essential for survival (can use glucose as carbon
source)
Induction increases expression 1000 fold making mutant identification
easy

Color changes using b-galactosidase enzyme (e.g., OPNG, X-gal) make
measurement of expression levels efficient
Coordinate repression and induction of three genes
revealed by studies of lactose-utilization mutants

Jacques Monod and Francois Jacob –
Pasteur Institute in Paris

Proposed Operon Theory of gene regulation
Single signal can simultaneously regulate expression
of several genes that are clustered together on a
chromosome and involve the same process
 Because genes are clustered, they are transcribed
together as single mRNA
 Clusters of genes are called Operons

Complementation Analysis of mutants identifies
lactose utilization genes
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Monod et al. isolated many Lac- mutants unable to
utilize lactose
Complementation analysis identified three genes
(lacZ, lacY, and lacA) in a tightly linked cluster
Fig. 16.5
Experimental evidence for repressor protein

Isolated mutant in lacI gene
Constitutive mutant – synthesized bgalactosidase and lac permease even in absence
of lactose (inducer)
 lacI must be a repressor – cells must need lacI
protein product to prevent expression of lacY
and lacZ in absence of inducer

PaJaMo experiment
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Fig. 16.6
lacI+, lacZ+ x lacI- lacZ- FInitial synthesis of bgalactosidase stops
Addition of inducer resumes
synthesis
Conclusion – initial lack of
repressor allows synthesis.
As lacI is transferred,
synthesis stops
Repressor stops
transcription by binding to
operator site near promoter
Inducer releases repressor to trigger
enzyme synthesis
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Addition of lactose inducer caused b –
galactosidase synthesis to continue
Conclusion: Inducer binds to repressor so
repressor can not bind to DNA
Allosteric effect - inducer bound to
promotor changes conformation of protein
so it can not bind to DNA
Repressor has binding domains for
operator and for the inducer
Fig. 16.7
Changes in the operator can also
affect repressor activity
Fig. 16.8
Proteins act in trans
DNA sites act only in cis

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Trans acting elements can diffuse through
cytoplasm and act at target DNA sites on
any DNA molecule in cell
Cis acting elements can only influence
expression of adjacent genes on same DNA
molecule
Three experiments elucidate cis and trans
acting elements using F’ plasmid
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
Insert Figure 16.9a
here
Fig. 16.9 a
Inducible synthesis
lacI+ gene encodes a
diffusible element that
acts in trans by
binding to any
operator it encounters
regardless of
chromosomal location
Noninducible

Insert Figure 16.9b
here
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Fig. 16.9 b
All operator sites (O+)
eventually occupied by
superrepressor
lacI supperrepressor can
not bind inducer
lacIs mutant encodes a
diffusible element that
binds to operator
regardless of chromosomal
location (trans acting
element)
Constitutive

Insert Figure 16.9c
here

Fig. 16.9 c
Presence of O+
plasmid does not
compensate for Oc
mutation on bacterial
chromosome
Operator is cis acting
element
The Operon Theory

The players
 lacz, lacY, lacZ genes that split lactose into glucose and galactose
 Promotor site to which RNA polymerase binds
 cis acting operator site
 trans-acting repressor that can bind to operator (encoded by lacI gene)
 Inducer that prevents repressor from binding to operator
Fig. 16.10 a
The Operon Theory
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Repression
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In absence of lactose, repressor binds to operator which
prevents transcription
Negative regulatory element
Fig. 16.10 b
The Operon Theory
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Induction
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Lactose present
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Allolactose binds to
repressor.
Repressor changes shape
and can not bind to
operator
RNA polymerase binds
to promotor and initiates
transcription of
polycistronic mRNA
Fig. 16.10 c
Positive control increases
transcription of lacZ, lacY, and lacA
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cAMP binds to
CRP (cAMP
receptor protein)
when glucose is low
CRP binds to
regulatory region
Enhances activity
of RNA polymerase
at lac promotor
Fig. 16.11
Some positive regulators increase
transcription of genes in only one pathway
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Fig. 16.12
AraC is a positive
regulator for all
arabinose genes
which break down
sugar arabinose
Loss of function
mutation results in
little or no
expression of genes
Molecular studies help fill in details
of control mechanisms
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Radioactive tag attached
to lac repressor
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Repressor from lacI+ cells
purified and mixed with
operator DNA, cosediment
occurred
Repressor from lacI+ mixed
with mutant operator DNA,
no cosediment occurred
Fig. 16.13
Many DNA-Binding proteins contain
a helix-turn-helix motif
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Fig. 16.14 a
Two α-helical regions
separated by a turn in
the protein structure
Helix-turn-helix motif
fits into major groove
of DNA
Most repressor
proteins
Specific amino acids in the a-helix determine
the binding specificity of repressor proteins

Hybrid 434-P22 repressor engineered to have amino acid
sequence that will bind to bacterial virus 434 and
bacteriophage P22
Fig. 16.14 b
Most regulatory proteins are
oligomeric
 More than one
binding domain
DNase footprint
identifies binding
region
DNase cannot
digest protein
covered sites
Fig. 16.15 a
The looping of DNA is a common
feature of regulatory proteins
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AraC acts as both a
repressor and activator

No arabinose
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Arabinose present
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Fig. 16.16
Binding to araO and
araI1 causes looping and
prevents RNA from
transcribing
AraC binds to araI1 and
araI2 bot not to araO. RNA
polymerase interacts with
araC at the araI sites and
transcribes genes
How regulatory proteins interact
with RNA polymerase
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Negative regulators (lac repressor)
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Physically block DNA-binding sites of RNA
polymerase
Positive regulators
Establish physical contact with RNA
polymerase enhancing enzyme’s ability to
initiate transcription
Using the lacZ gene as a reporter of
gene expression
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Reporter gene – protein encoding gene
whose expression in the cell is quantifiable
by techniques of protein detection.
Fusion of reporter gene to cis acting
regulatory regions allows assessment gene
activity by monitoring amount of reporter
gene product
Fusion used to perform genetic studies of the
regulatory region of gene X
Fig. 16.18 a
Creating a
collection of
lacZ
insertions in
the
chromosome
Fig. 16.18 b
Use of a fusion to
overproduce a gene
product
Fig. 16.18 c
The attenuation of gene expression: Fine tuning of the trp
operon through termination of transcription

The presence of tryptophan activates a
repressor of the trp operon

trpR gene produces repressor

Corepressor – tryptophan binds to trp repressor
allowing it to bind to operator DNA and inhibit
transcription
Termination of transcription fine
tunes regulation of trp operon

trpR- mutants are not constitutive
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Repressor independent change in trp expression
Two alternative transcripts lead to different
transcriptional outcomes

Leader sequence can fold in two different stable
conformations
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Tryptophan present – ribosome moves quickly past codons in
leader allowing stem-loop to form terminating transcription
Tryptophan absent – ribosome stalls allowing normal stem loop
structure to form and transcription proceeds normally
Global regulatory mechanisms coordinate the
expression of many genes
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Normal sigma factor (s70) binds to RNA polymerase and
recognizes sequence in promoter to initiate transcription
Heat shock disables s70
Product of rpoH gene, s32 binds to sequence in promoter of
heat shock genes when heat stressed and starts
transcription
Fig. 16.21 a
Factors influencing increase in s32
activity after heat shock
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Increase in transcription of the rpoH gene
Increase in the translation of s32 mRNA
stemming from greater stability of rpoH mRNA
Increase in the stability and activity of the s32
protein. Chaperones DnaJ/K bind and inhibit s32
under normal conditions. At high temperature,
binding to s32 does not occur and more s32 is free
to associate with RNA polymerase.
Inactivity of s70 decreases competition with s32 to
form RNA polymerase holoenzyme
What enables transcription of s32
during heat shock?
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Normal temperatures, rpoH gene (encodes
s32) has promoter sequence recognized by s70
which starts transcription
High temperatures (no s70) a different promoter
sequence of the rpoH gene is recognized by a
different sigma factor, s24
Summary
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E. coli’s heat shock response is controlled by
alternative sigma factors that recognize different
promoter sequences
Alternative sigma factors bind to RNA polymerase
as temperatures change to start transcription of
heat shock proteins
The induction of alternative sigma factors that
recognize different promoter sequences serve as
global control regulatory mechanisms in E. coli
and many other bacteria
Microarrays – a tool for uncovering
changes in gene expression

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Cellular responses to global environmental
changes can be measured by microarray
analysis of mRNA isolated from cultures
grown in different environmental conditions
Comparisons of wild-type cultures with
strains containing mutations in key
regulatory regions help identify genes and
regulatory elements involved in response to
specific environmental changes
Fig. 1.13
Regulation of Virulence Genes in
V. cholerae
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Bacterial agents of cholera sense changes in
environment and transmit signals to
regulators that initiate, enhance, diminish,
or repress expression of various genes.
Three regulatory proteins – ToxR, ToxS,
and ToxT – turn on the genes for virulence
Experiments generate model for regulation of
virulence genes in V. cholerae
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Cloned two genes encoding subunits of
cholera toxin: ctxA and ctxB
Made ctxA-lacZ reporter gene fusion
Created vector library of V. cholerae
genomic DNA
Used E. coli to perform genetic
manipulations

Isolated a gene that regulates expression of ctx operon
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Transformed E. coli containing ctx-lacZ construct with clones
containing V. cholerae DNA
Clones that contain a positive regulator should turn on ctx-lacZ
construct
Identified ToxR, a transmembrane protein
Identified ToxS, helps ToxR form dimers which helps it bind to DNA
What genes does ToxR regulate?
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Gene fusions created to constitutive promoter
Fusion introduced into strains of V. cholerae with lacZ randomly inserted
around the genome
Identified intermediate regulator gene ToxT, a transcriptional activator
that binds to promoters of many genes, including ctx
ToxR/S or ToxT can activate the ctx genes that produce toxin
ToxT alone activates additional virulence genes which encode pili and
other proteins
Transcription of ToxT is regulated by ToxR/S
Fig. 16.22
Unanswered Questions Remain
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Why is there a cascade (ToxR and ToxT) of
regulatory factors?
What DNA sequence in the promoters does
ToxR recognize?
What is the signal that’s makes the cholera
bacteria start to colonize the small
intestine?
How does ToxR regulatory protein find
binding sites on the chromosome?