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PART V

How Genes Are Regulated

Gene Regulation in
Prokaryotes

CHAPTER OUTLINE

Overview of Prokaryotic Gene Regulation


The Regulation of Gene Transcription
Attenuation of Gene Expression: Termination of Transcription
Global Regulatory Mechanisms
Comprehensive Example: The Regulation of Virulence Genes in V. cholerae
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15

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

Two key aspects of the life of a unicellular


prokaryote
1)

direct contact with their external environment

2)

the ability to respond to changes in that environment by changes in gene


expression.
This coordinated control of gene expression
in a bacterial cell is an example of prokaryotic
gene regulation.

RNA polymerase participates in all three phases of


transcription
Initiation core RNA polymerase plus sigma () factor
Core has four subunits: two alpha (), one beta (), one beta prime (')
DNA is unwound and polymerization begins

Elongation core RNA polymerase without factor


Continues until RNA polymerase recognizes termination signal

Termination two kinds in bacteria


Rho-dependent Rho () protein binds to RNA and removes it from RNA
polymerase
Rho-independent 20 nt sequence in RNA forms stem-loop

Role of RNA polymerase in initiation and elongation phases


of transcription

Fig. 15.2

Two kinds of transcription termination in bacteria

Fig. 15.2 (cont)

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

In prokaryotes, RNA polymerase is the key enzyme in transcription.

Translation begins before transcription ends in these organisms,


and regulation of gene expression can occur at many different
points in this process.

Utilization of lactose by E. coli provides a model


system of gene regulation
The binding of regulatory proteins to DNA targets controls
transcription.
either inhibits or enhances the effectiveness of RNA polymerase in
initiating transcription

the inhibition of RNA polymerase activity as negative regulation


the enhancement of RNA polymerase activity as positive
regulation.

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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
-Galactosidase (-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 -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

E. coli grown in
medium containing
both glucose and
lactose,
they prefer glucose.

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 -gal (color change)
Lactose induces a 1000-fold increase in -gal activity
Detection and characterization of hundreds of lac mutants defective in
lactose utilization

13

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

14

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

=alloctase

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

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Jacob and Monod defined the roles of the lac


genes by genetic analysis of many lacI mutants
genetic analysis to develop a molecular model explaining
how environmental changes could provoke changes in
gene activity
the operon theory of gene regulation, Monod and his
collaborators isolated many different Lac mutants, that
is, bacterial cells unable to utilize lactose.

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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 -galactosidase
lacY encodes permease
lacA encodes transacetylase (adds an acetyl (CH3CO) group to lactose and other b-galactoside
sugars).
Most studies focused on lacZ and lacY

Constitutive expression of -galactosidase and permease was caused by mutations in the lacI gene
Constitutive mutants (lacI) express the enzymes in the absence and presence of inducer
(irrespective of environmental conditions.)

The existence of these constitutive mutants suggested that lacI encodes a negative regulator, or
repressor.

a mutation in the lacI gene generates a defect in the repressor protein that prevents it from carrying out
this negative regulatory function

19

The PaJaMo experiment provided evidence that


lacI encodes a repressor
lacI+ lacZ+ DNA transferred into
lacI lacZ cells
-gal levels increased initially
-gal levels decreased as
repressor accumulated
-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

20

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

21

If the repressor protein interacts with both the operator and the
inducer, what outcome would you predict for mutations that disrupt
one of these interactions without affecting the other

????

22

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

23

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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How can one distinguish the constitutive operator ( lacO


c ) mutants from the previously described constitutive
lacI mutants, considering that both prevent repression?
The answer is found in a cis / trans test.

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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

31

Summary of negative control


The lacOC constitutive operator has the same
effect as the lacI constitutive mutant.
These can be distinguished by the cis/trans test:
An altered protein acts in trans, whereas an altered
operator sequence acts in cis.

Experiments in the 1970s verifi ed that the lac


genes are transcribed as a cluster ( polysistronic)
that has a single promoter.

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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 (cyclic adenosine monophosphate) binds to cAMP receptor protein (CRP)
CRP-cAMP is a positive regulator of lac transcription

33

Positive regulation by CRPcAMP (cyclic adenosine monophosphate)


Catabolite repression overall effect of glucose is to prevent lac gene
expression even in the presence of lactose!
The binding of cAMP to CRP
enables CRP to bind to DNA in
the regulatory region of the lac
operon, and this DNA binding
of CRP increases the ability of
RNA polymerase to transcribe
the lac genes.

Fig. 15.15

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Positive regulation by CRPcAMP (cyclic


adenosine monophosphate)

The CRPcAMP complex also increases transcription in several


other catabolic gene systems,
the gal operon (whose protein products help break down the
sugar galactose)
the ara operon (contributing to the breakdown of the sugar
arabinose).

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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

The loss of function of a regulatory protein results in little or no expression of


the regulated genes, the protein must be a positive regulator.
By contrast, loss of function of a negative regulator causes constitutive
production of the operons gene products.

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AraC is a positive regulator

Fig. 15.16

37

How DNA-binding proteins control initiation of operon


transcription: A summary
The initiation of transcription by RNA polymerase is under the control of
regulatory genes whose products bind to specifi c DNA sequences in the
vicinity of the promoter.
negative regulatory proteins prevents the initiation of transcription;
the binding of positive regulators assists the initiation of transcription.

lac operon depends on at least two proteins: the repressor (a


negative regulator) and CRP (a positive regulator).
Transcription of the arabinose operon, for example, which is induced
in the presence of arabinose, receivesa boost from two positive
regulators: the CRPcAMP complex and AraC.

38

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 -helices of lac repressor are
required for binding to lacO (Fig. 15.19)
DNA sequences to which negative and positive regulators bind have a twofold 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


Radioactive tag is attached to the lac
repressor protein so it can be
followed in the experiment.
(a) When repressor protein from
lacl+ cells was purified and mixed
with DNA containing the lac operator
(on bacterial virus DNA), the protein
cosedimented with the DNA.
(b) When wild-type repressor was
mixed with DNA containing
a mutant operator site, no
radioactivity sedimented with the
DNA.
Fig. 15.17

40

DNA recognition sequences by helix-turn-helix (HTH) motif


A protein with an HTH motif has two -helical
regions separated by a turn in the protein
The HTH motif fits into the major groove of DNA
One of the -helices recognizes a specific DNA
sequence
each HTH has a specifi city for DNA binding
based on its sequence of amino acids

Fig. 15.18

41

Changing amino acids in recognition sequence of a


repressor protein

434 repressor binds to an operator in the DNA of the 434 virus that has integrated into the bacterial
genome and prevents transcription and production of viral particles.

P22 repressor binds to an operator in the DNA of the P22 virus

Amino acid sequences in the -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

42

The HTH motif is found in hundreds of DNA-binding proteins.


more than 20 different DNA-binding proteins in bacteria are very
similar to the LacI repressor, not only in the HTH DNA-binding
domain but throughout much of the protein.
This group of repressors is known as the LacI repressor family of
proteins
The uniqueness of their DNA-recognition regions means that they
interact with different operators to regulate different groups of
genes.

43

DNase footprint shows where proteins bind


DNase footprint establishes the
region to which a protein binds.
A partial digestion with DNase I
produces a series of fragments. If
a protein is bound to DNA,
DNase cannot digest at sites
covered by the protein.
Gel electrophoresis of digested
products shows which products
were not generated and indicates
where the protein binds.

44

5'TGTGAGTTAGCTCACA3'
3'ACACTCAATCGAGTGT5'
many of the DNA sequences to which a negative or positive regulator
protein binds exhibit rotational symmetry;
that is, their two DNA strands have an almost identical sequence when read
in the 5-to-3 direction on both strands
REGULATORY PROTEINS ARE OLIGOMERS COMPOSED OF TWO TO
FOUR POLYPEPTIDE SUBUNITS
oligomeric protein can bind in a genes regulatory region as clusters,
many contacts can be established between the protein and the regulatory region.

Increasing the stability of protein-DNA interactions,


necessary to maintain repression or activate transcription.

45

CRPcAMP binds as a dimer to a regulatory region


CRP-binding sites have a two-fold rotational symmetry
CRP protein binds as a dimer
CRP-binding site consists of two recognition sequences, one for each subunit of
the CRP dimer

one monomer of CRP


binding to each side of the
sequence

Fig. 15.21

46

lac repressor tetramer binds to two sites


lac repressor is a tetramer, with each subunit containing a DNAbinding 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

47

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

Fig. 15.23

48

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)

49

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

50

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

51

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 (promoters and operators)

from other genes (Fig. 15.26)


Make mutant gene Xs control region and check for function
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)
Inducible expression of the foreign gene controlled by IPTG

52

Measuring gene expression


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 -galactosidase
Specific regulatory sites can be identified by constructing and testing mutations in the
test regions of gene X

Fig. 15.26

53
Identifying sets of genes regulated
by the same stimulus

lacZ introduced into a population


of E. coli cells.
Researchers identified a set of
genes activated by exposure to
DNA-damaging agents by this
method.

Fig. 15.27

Controlling gene expression


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

Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15

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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

56

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

57

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

58

Sigma factor () recognition sequences


Normal, housekeeping sigma factor is 70
Active under normal physiological conditions, but is inactivated by heat
shock

rpoH genes encodes 32, an alternative sigma factor


Heat shock inducible genes have promoters that are recognized by 32
32 is resistant to inactivation by heat shock

Fig. 15.31

59

Factors influencing increase in 32 activity


after heat-shock treatment
Increased transcription of rpoH gene
Increased translation of 32 mRNA because of increased stability of rpoH
mRNA
Increased stability and activity of 32 protein
No longer inhibited by chaperones DnaJ/K
Chaperones DnaJ/K bind to and inhibit 32 under normal physiological
condition.

The inactivity of 70 at high temperatures


No competition from 70 because it is removed by degradation

60

Alternate sigma factor in the heat-shock response


At normal temperatures, promoter for rpoH gene is recognized by 70
After heat shock, 70 is degraded and transcription of the gene for 24 is
increased
24 recognizes a different promoter sequence at rpoH
Increased expression of 32 causes transcription of several genes

Fig. 15.32

61

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

62

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

63

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

64

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

65

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 -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

66

Model for how V. cholerae regulates genes for


virulence

Fig. 15.34

67

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