Gene Regulation in

Prokaryotes

Outline of Chapter 16

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

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. colis

response to heat shock is an example of the
bacterial ability to coordinate the expression of
different sets of genes dispersed around the
chromosome.

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)

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

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 Us signals release of RNA
polymerase

Fig. 16.2

Translation in prokaryotes starts before transcription

ends

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

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

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

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 -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

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 galactosidase 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

Fig. 16.6

lacI+, lacZ+ x lacI- lacZ- FInitial synthesis of galactosidase 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

Addition of lactose inducer caused

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

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

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

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

Repression

In absence of lactose, repressor binds to operator which

prevents transcription
Negative regulatory element
Fig. 16.10 b

The Operon Theory

Induction

Lactose present

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

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

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

Radioactive tag attached

to lac repressor

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

Fig. 16.14 a

Two -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

AraC acts as both a

repressor and activator

No arabinose

Arabinose present

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

Negative regulators (lac repressor)

Physically block DNA-binding sites of RNA

polymerase

Positive regulators
Establish physical contact with RNA
polymerase enhancing enzymes ability to
initiate transcription

Using the lacZ gene as a reporter of

gene expression

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

Repressor independent change in trp expression

Two alternative transcripts lead to different

transcriptional outcomes

Leader sequence can fold in two different stable

conformations

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

Normal sigma factor ( 70) binds to RNA polymerase and

recognizes sequence in promoter to initiate transcription

Heat shock disables 70

Product of rpoH gene, 32 binds to sequence in promoter of
heat shock genes when heat stressed and starts transcription
Fig. 16.21 a

Factors influencing increase in 32

activity after heat shock

Increase in transcription of the rpoH gene

Increase in the translation of 32 mRNA

stemming from greater stability of rpoH mRNA

Increase in the stability and activity of the 32
protein. Chaperones DnaJ/K bind and inhibit 32
under normal conditions. At high temperature,
binding to 32 does not occur and more 32 is free
to associate with RNA polymerase.
Inactivity of 70 decreases competition with 32 to
form RNA polymerase holoenzyme

What enables transcription of 32

during heat shock?

Normal temperatures, rpoH gene (encodes

32) has promoter sequence recognized by 70
which starts transcription
High temperatures (no 70) a different promoter
sequence of the rpoH gene is recognized by a
different sigma factor, 24

Summary

E. colis 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

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

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

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

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?

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

Why is there a cascade (ToxR and ToxT) of

regulatory factors?
What DNA sequence in the promoters does
ToxR recognize?
What is the signal thats makes the cholera
bacteria start to colonize the small
intestine?
How does ToxR regulatory protein find
binding sites on the chromosome?