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Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 14
GENE REGULATION IN BACTERIA
AND BACTERIOPHAGES
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INTRODUCTION

The term gene regulation means that the level of
gene expression can vary under different conditions

Genes that are unregulated are termed constitutive



They have essentially constant levels of expression
Frequently, constitutive genes encode proteins that are
necessary for the survival of the organism
The benefit of regulating genes is that encoded
proteins will be produced only when required
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14-2
INTRODUCTION

Gene regulation is important for cellular processes
such as




1. Metabolism
2. Response to environmental stress
3. Cell division
Regulation can occur at any of the points on the
pathway to gene expression

Refer to Figure 14.1
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14-3
Figure 14.1
14-4
14.1 TRANSCRIPTIONAL
REGULATION

The most common way to regulate gene expression in
bacteria is at the transcriptional level

The rate of RNA synthesis can be increased or decreased

Transcriptional regulation involves the actions of two main
types of regulatory proteins
 Repressors  Bind to DNA and inhibit transcription
 Activators  Bind to DNA and increase transcription

Negative control refers to transcriptional regulation by
repressor proteins
 Positive control to regulation by activator proteins
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14-5

Small effector molecules affect transcription regulation


However, these bind to regulatory proteins and not to DNA directly
In some cases, the presence of a small effector molecule
may increase transcription


These molecules are termed inducers
They function in two ways




Bind activators and cause them to bind to DNA
Bind repressors and prevent them from binding to DNA
Genes that are regulated in this manner are termed inducible
In other cases, the presence of a small effector molecule
may inhibit transcription



Corepressors bind to repressors and cause them to bind to DNA
Inhibitors bind to activators and prevent them from binding to DNA
Genes that are regulated in this manner are termed repressible
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14-6

Regulatory proteins have
two binding sites


Figure 14.2
One for a small effector
molecule
The other for DNA
14-7
Figure 14.2
14-8
The Phenomenon of Enzyme
Adaptation

At the turn of the 20th century, scientists made the
following observation



A particular enzyme appears in the cell only after the cell
has been exposed to the enzyme’s substrate
This observation became known as enzyme adaptation
François Jacob and Jacques Monod at the Pasteur
Institute in Paris were interested in this phenomenon

They focused their attention on lactose metabolism in
E. coli to investigate this problem
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14-9
The lac Operon


An operon is a regulatory unit consisting of a few
structural genes under the control of one promoter

It encodes polycistronic mRNA that contains the coding
sequence for two or more structural genes

This allows a bacterium to coordinately regulate a group
of genes that encode proteins with a common function
An operon contains several different regions

Promoter; terminator; structural genes; operator
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14-10


Figure 14.3a shows the organization and transcriptional
regulation of the lac operon genes
There are two distinct transcriptional units
 1. The actual lac operon

a. DNA elements




Promoter  Binds RNA polymerase
Operator  Binds the lac repressor protein
CAP site  Binds the Catabolite Activator Protein (CAP)
b. Structural genes



lacZ  Encodes b-galactosidase
 Enzymatically cleaves lactose and lactose analogues
 Also converts lactose into allolactose (an isomer)
lacY  Encodes lactose permease
 Membrane protein required for transport of lactose and analogues
lacA  Encodes transacetylase
 Covalently modifies lactose and analogues
 Its functional necessity remains unclear
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14-11


Figure 14.3a shows the organization and transcriptional
regulation of the lac operon genes
There are two distinct transcriptional units

2. The lacI gene

Not considered part of the lac operon

Has its own promoter, the i promoter

Constitutively expressed at fairly low levels

Encodes the lac repressor

The lac repressor protein functions as a tetramer

Only a small amount of protein is needed to repress the lac operon

There is usually ten tetramer proteins per cell
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14-12
Figure 14.3
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14-13
The lac Operon Is Regulated By
a Repressor Protein

The lac operon can be transcriptionally regulated



1. By a repressor protein
2. By an activator protein
The first method is an inducible, negative control
mechanism


It involves the lac repressor protein
The inducer is allolactose


It binds to the lac repressor and inactivates it
Refer to Figure 14.4
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14-14
RNA pol
cannot access
the promoter
Constitutive
expression
The lac operon is now
repressed
Therefore no allolactose
Figure 14.4
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14-15
Translation
The lac operon is now
induced
The conformation of the
repressor is now altered
Repressor can no longer
bind to operator
Some gets converted to allolactose
Figure 14.4
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14-16
Repressor does not completely
inhibit transcription
So very small amounts of the
enzymes are made
Figure 14.5
The cycle of lac operon induction and repression
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14-17
Experiment 14A: The lacI Gene
Encodes a Repressor Protein

In the 1950s, Jacob and Monod, and their colleague
Arthur Pardee, had identified a few rare mutant
strains of bacteria with abnormal lactose adaptation

One type of mutant involved a defect in the lacI gene



It was designated lacI–
It resulted in the constitutive expression of the lac operon
even in the absence of lactose
The lacI– mutations mapped very close to the lac operon
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14-18

Jacob, Monod and Pardee proposed two different
functions for the lacI gene
Figure 14.6

Jacob, Monod and Pardee applied a genetic
approach to distinguish between the two hypotheses
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14-19



They used bacterial conjugation methods to
introduce different portions of the lac operon into
different strains
They identified F’ factors (plasmids) that carried
portions of the lac operon
For example: Consider an F’ factor that carries the
lacI gene

Bacteria that receive this will have two copies of the lacI
gene


One on the chromosome and the other on the F’ factor
These are called merozygotes, or partial diploids
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14-20


Merozygotes were instrumental in allowing Jacob
et al to elucidate the function of the lacI gene
There are two key points

1. The two lacI genes in a merozygote may be different
alleles



lacI– on the chromosome
lacI+ on the F’ factor
2. Genes on the F’ factor are not physically connected to
those on the bacterial chromosome

If hypothesis 1 is correct


The repressor protein produced from the F’ factor can diffuse and
regulate the lac operon on the bacterial chromosome
If hypothesis 2 is correct

The binding site on the F’ factor cannot affect the lac operon on the
bacterial chromosome, because they are not physically adjacent
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14-21
The Hypothesis

The lacI gene either/or


1. Encodes a regulatory protein (the lac repressor)
that can diffuse throughout the cell
2. Acts as a binding site for a repressor protein

Thus it can only act on a physically connected lac operon
Testing the Hypothesis

Refer to Figure 14.7
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14-22
Figure 14.7
14-23
Figure 14.7
14-24
Figure 14.7
14-25
The Data
Strain
Addition of lactose
Amount of b-galactosidase
(percentage of parent strain)
Mutant
No
100%
Mutant
Yes
100%
Merozygote
No
<1%
Merozygote
Yes
220%
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14-26
Interpreting the Data
Strain
Addition of lactose
Expected result because of
constitutive expression in the
lacI– strain
Amount of b-galactosidase
(percentage of parent strain)
Mutant
No
100%
Mutant
Yes
100%
Merozygote
No
<1%
Merozygote
Yes
220%
In the presence of lactose, both lac
operons are induced, yielding a
higher level of enzyme activity
In the absence of
lactose, both lac
operons are repressed
This result is consistent with hypothesis 1
The lacI gene codes for a repressor protein that can
diffuse throughout the cell and bind to any lac operon
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14-27
Interpreting the Data

The interaction between regulatory proteins and DNA
sequences have led to two definitions

1. Trans-effect




Genetic regulation that can occur even though DNA segments are
not physically adjacent
Mediated by genes that encode regulatory proteins
Example: The action of the lac repressor on the lac operon
2. Cis-effect or cis-acting element



A DNA sequence that must be adjacent to the gene(s) it regulates
Mediated by sequences that bind regulatory proteins
Example: The lac operator
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14-28
Interpreting the Data

Table 14.1 summarizes the effects of lacI gene
mutations versus lacO (operator) in merozygotes

Overall

A mutation in a trans-acting factor is complemented by
the introduction of a second gene with a normal function

However, a mutation in a cis-acting element is not!
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14-29
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14-30
The lac Operon Is Also Regulated
By an Activator Protein

The lac operon can be transcriptionally regulated in
a second way, known as catabolite repression

When exposed to both lactose and glucose



E. coli uses glucose first, and catabolite repression
prevents the use of lactose
When glucose is depleted, catabolite repression is
alleviated, and the lac operon is expressed
The sequential use of two sugars by a bacterium is
termed diauxic growth
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14-31
The lac Operon Is Also Regulated
By an Activator Protein

The small effector molecule in catabolite repression
is not glucose

This form of genetic regulation involves a small
molecule, cyclic AMP (cAMP)

It is produced from ATP via the enzyme adenylyl cyclase

cAMP binds an activator protein known as the Catabolite
Activator Protein (CAP)

Also termed the cyclic AMP receptor protein (CRP)
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14-32
The lac Operon Is Also Regulated
By an Activator Protein

The cAMP-CAP complex is an example of genetic
regulation that is inducible and under positive control


The cAMP-CAP complex binds to the CAP site near the
lac promoter and increases transcription
In the presence of glucose, the enzyme adenylyl
cyclase is inhibited

This decreases the levels of cAMP in the cell

Therefore, cAMP is no longer available to bind CAP
 Transcription rate decreases
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14-33
(b) Lactose but no cAMP
Figure 14.8
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14-34
Figure 14.8
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14-35
The lac Operon Has Three
Operator Sites for the lac Repressor

Detailed genetic and crystallographic studies have
shown that the binding of the lac repressor is more
complex than originally thought

In all, three operator sites have been discovered




O1  Next to the promoter
O2  Downstream in the lacZ coding region
O3  Slightly upstream of the CAP site
Refer to Figure 14.9
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14-36
Repression is 1,300 fold
Therefore, transcription is 1/1,300
the level when lactose is present
No repression
ie: Constitutive expression
Figure 14.9
The identification of three lac operator sites
14-37

The results of Figure 14.9 supported the hypothesis
that the lac repressor must bind to two of the three
operators to cause repression

It can bind to O1 and O2 , or to O1 and O3



If either O2 or O3 is missing maximal repression is not
achieved
Binding of the lac repressor to two operator sites
requires that the DNA form a loop

A loop in the DNA brings the operator sites closer together


But not O2 and O3
This facilitates the binding of the repressor protein
Refer to Figure 4.14
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14-38
Each repressor
dimer binds to one
operator site
Each repressor
dimer binds to one
operator site
Figure 14.10
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14-39
The ara Operon

Another operon in E. coli that is involved in sugar
metabolism is the ara (arabinose) operon

It contains

Three structural genes involved in arabinose metabolism




These are designated araB, araA and araD
A single promoter, PBAD
A CAP site, which binds the catabolite activator protein
Refer to Figure 14.11
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14-40

The araC gene is adjacent to the ara operon



It has its own promoter, PC
It encodes a regulatory protein, AraC
AraC can bind to three different operator sites

Designated araI, araO1 and araO2
The AraC protein can act as either a
negative or positive regulator of
transcription
Depending on whether or not
arabinose is present
Figure 14.11
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14-41

AraC protein binds to all three operators

AraC dimer bound to araO1 inhibits transcription of the araC gene


This keeps AraC protein levels fairly low
AraC monomers bound to araO2 and araI repress the ara operon

They bind to each other (via looped DNA), and block RNA pol access to PBAD
araO1
Figure 14.12
14-42

Arabinose binds to the AraC proteins

The interaction betweem the AraC proteins at the araO2 and araI is broken


This breaks the DNA loop
Another, AraC protein binds to araI

This AraC dimer at araI activates transcription
CAP-cAMP activation occurs
when glucose levels are low
Figure 14.12
14-43
The trp Operon

The trp operon (pronounced “trip”) is involved in the
biosynthesis of the amino acid tryptophan

The genes trpE, trpD, trpC, trpB and trpA encode
enzymes involved in tryptophan biosynthesis

The genes trpR and trpL are involved in regulation

trpR  Encodes the trp repressor protein


Functions in repression
trpL  Encodes a short peptide called the Leader peptide

Functions in attenuation
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14-44
RNA pol can bind
to the promoter
Cannot bind to
the operator site
Figure 14.13 Organization of the trp operon and regulation via the trp
repressor protein
14-45
Figure 14.13 Organization of the trp operon and regulation via the trp
repressor protein
14-46
Another mechanism
of regulation
Figure 14.13 Organization of the trp operon and regulation via the trp
repressor protein
14-47

Attenuation occurs in bacteria because of the coupling of
transcription and translation

During attenuation, transcription actually begins but it is
terminated before the entire mRNA is made




A segment of DNA, termed the attenuator, is important in facilitating
this termination
In the case of the trp operon, transcription terminates shortly past the
trpL region (Figure 14.13c)
Thus attenuation inhibits the further production of tryptophan
The segment of trp operon immediately downstream from
the operator site plays a critical role in attenuation

The first gene in the trp operon is trpL


It encodes a short peptide termed the Leader peptide
Refer to Figure 14.14
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14-48


Region 2 is complementary to regions 1 and 3
Region 3 is complementary to regions 2 and 4
 Therefore several stem-loops structures are possible
The 3-4 stem loop is
followed by a sequence
of Uracils
It acts as an intrinsic
(r-independent) terminator
These two codons provide a way
to sense if there is sufficient
tryptophan for translation
Figure 14.14 Sequence of the trpL mRNA produced during attenuation
14-49

Therefore, the formation of the 3-4 stem-loop
causes RNA pol to terminate transcription at the
end of the trpL gene

Conditions that favor the formation of the 3-4
stem-loop rely on the translation of the trpL mRNA

There are three possible scenarios

1. No translation
2. Low levels of tryptophan
3. High levels of tryptophan

Refer to Figure 14.15


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14-50
Transcription terminates
just past the trpL gene
Most stable form of
mRNA occurs
Therefore no coupling of
transcription and translation
Figure 14.15 Possible stem-loop structures formed from trpL mRNA under
different conditions of translation
14-51
Region 1 is blocked
3-4 stem-loop
does not form
RNA pol transcribes
rest of operon
Insufficient amounts
of tRNAtrp
Figure 14.15 Possible stem-loop structures formed from trpL mRNA under
different conditions of translation
14-52
Sufficient amounts of tRNAtrp
3-4 stem-loop forms
Translation of the trpL mRNA
progresses until stop codon
RNA polymerase pauses
Transcription
terminates
Region 2 cannot base pair
with any other region
Figure 14.15 Possible stem-loop structures formed from trpL mRNA under
different conditions of translation
14-53
Inducible vs Repressible Regulation

The study of many operons revealed a general trend
concerning inducible versus repressible regulation

Operons involved in catabolism (ie. breakdown of a
substance) are typically inducible


The substance to be broken down (or a related compound) acts
as the inducer
Operons involved in anabolism (ie. biosynthesis of a
substance) are typically repressible

The inhibitor or corepressor is the small molecule that is the
product of the operon
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14-54
14.2 TRANSLATIONAL AND
POSTTRANSLATIONAL
REGULATION

Genetic regulation in bacteria is exercised
predominantly at the level of transcription


However, there are many examples of regulation that occur
at a later stage in gene expression
For example, regulation of gene expression can be


Translational
Posttranslational
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14-55
Translational Regulation

For some bacterial genes, the translation of mRNA
is regulated by the binding of proteins

A translational regulatory protein recognizes
sequences within the mRNA

In most cases, these proteins act to inhibit
translation

These are known as translational repressors
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14-56
Translational Regulation

Translational repressors inhibit translation in one of
two ways

1. Binding next to the Shine-Dalgarno sequence and/or
the start codon


2. Binding outside the Shine-Dalgarno/start codon region


This will sterically hinder the ribosome from initiating translation
They stabilize an mRNA secondary structure that prevents initiation
Translational repression is also found in eukaryotes
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14-57
Translational Regulation

A second way to regulate translation is via the
synthesis of antisense RNA


An RNA strand that is complementary to mRNA
Consider, for example, the trait of osmoregulation

The ability to control the amount of water inside the cell

The protein ompF in E. coli is important in osmoregulation


This outer membrane protein is encoded by the ompF gene
OmpF protein is preferentially produced at low osmolarity

At high osmolarity its synthesis is decreased
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14-58

The expression of another gene, termed micF, is responsible
for inhibiting the ompF gene at high osmolarity


micF RNA does not code for a protein
It is, however, complementary to ompF mRNA
 It is thus termed antisense RNA
Figure 14.16
Its translation is now blocked
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14-59
Posttranslational Regulation

A common mechanism to regulate the activity of
metabolic enzymes is feedback inhibition

The final product in a pathway often can inhibit the
an enzyme that acts early in the pathway

Refer to Figure 14.17
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14-60

Enzyme 1 is an allosteric enzyme,
with two different binding sites


Catalytic site  binds substrate
Regulatory site  binds final
product of the pathway

If the concentration of product 3
becomes high

It will bind to enzyme 1

Thereby inhibiting its ability to
convert substrate 1 into product 1
Figure 14.17
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14-61
Posttranslational Regulation


A second strategy to control the function of proteins
is by the covalent modification of their structure
Some modifications are irreversible



Proteolytic processing
Attachment of prosthetic groups, sugars, or lipids
Other modifications are reversible and transiently
affect protein function



Phosphorylation (–PO4)
Acetylation (–COCH3)
Methylation (–CH3)
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14-62
14.3 GENE REGULATION IN THE
BACTERIOPHAGE LIFE CYCLE

Bacteriophages are viruses that infect bacteria


The structural genes of bacteriophages are often in
an operon arrangement


Their study has greatly advanced our basic knowledge of
genetic regulation
Like bacterial operons, phage operons can be controlled
by repressor proteins or activator proteins
To understand how this works, we will examine the
two life cycles of phage l (lambda)
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14-63
Life Cycles of Phage l


Phage l can bind to the surface of a bacterium and
inject its genetic material into the bacterial cytoplasm
The phage will then proceed along only one of two
alternative life cycles



Lytic cycle
Lysogenic cycle
Let’s review Figure 6.9
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14-64
This process is
termed
induction
It will undergo
the lytic cycle
Prophage can
exist in a dormant
state for a long
time
Virulent phages only
undergo a lytic cycle
Figure 6.9
Temperate phages can
follow both cycles
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14-65

Figure 14.18 shows the genome of phage l

Inside the viral head, phage l DNA is linear


After injection into the bacterium, the two ends attach covalently to
each other forming a circle
The organization of the genes within this circular structure
reflects the two alternative life cycles of the virus

The genes in the top center are transcribed very soon after infection,
at the beginning of either life cycle

The pattern of their expression determines which of the two cycles prevails

The genes on the left side of the viral genome encode proteins that are
responsible for the lysogenic infection

The genes on the right side of the viral genome encode proteins that
are responsible for the lytic infection
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14-66
Figure 14.18
14-67
Life Cycles of Phage l

So how is the decision made between the lytic and
lysogenic cycles?

The choice depends on the actions of several
genetic regulatory proteins

The process is quite detailed


It involves a series of intricate steps in which these
proteins bind to several different sites in the l genome
Refer to Figure 14.19
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14-68
The N protein is an antiterminator
Encoding two
proteins: N and cro
It binds to RNA polymerase and
prevents transcriptional termination
cIII gene encodes
a protein that
helps stabilize the
cII activator
protein
Figure 14.19
cII gene encodes an
activator protein
The O and P genes
encode enzymes needed
to initiate DNA synthesis
The Q gene encodes
another antiterminator
needed for the lytic cycle
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cII/cIII activates transcription from PI and PRE
PRE = Promoter for l Repressor during
Establishment of lysogenic cycle
int gene encodes the protein integrase, which
integrates l DNA into the bacterial chromosome
The l repressor binds to operators that
are adjacent to PR and PL
It thus inhibits the expression of genes
required for the lytic cycle
The l repressor also activates PRM
This is sufficient to make enough l
Repressor to Maintain the lysogenic cycle
Figure 14.19
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The cro protein binds to two operators OR and OL
Binding to OL inhibits transcription from PL
Binding to OR has several effects
1.
It inhibits transcription from PRM in the
leftward direction
•This prevents the expression of the cI
gene which encodes the l repressor
2.
It allows a low level of transcription from
PR in the rightward direction
• This enables the transcription of the O,
P and Q genes
The O and P proteins are necessary
for the replication of l DNA
Figure 14.19
The Q protein is an antiterminator that
permits transcription through another
promoter, PR’
PR’ controls a very large operon that
encodes the proteins necessary for the
assembly of the phage coat, packaging
of the DNA and lysis of the bacterial cell
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Cellular Proteases Influence the
Choice Between the Two Cycles

The activity of the cII protein plays a key role in
directing l to the lysogenic or lytic cycle

The cII protein is easily degraded by cellular
proteases produced by E. coli

Whether or not these proteases are produced
depends on the environmental conditions
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14-72

If the growth conditions are very favorable, the
intracellular levels of the proteases are high

The cII protein tends to be degraded


Instead, the cro protein slowly accumulates to high levels



Therefore, PRE cannot be activated and the l repressor is not made
The binding of the cro protein to OR prevents transcription of the l
repressor from PRM
At the same time, the cro protein allows the lytic cycle to proceed
Thus, environmental conditions that are favorable for
growth promote the lytic cycle

This makes sense because a sufficient supply of nutrients
is necessary to synthesize new bacteriophages
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14-73

If the nutrients are limiting (starvation conditions),
the cellular proteases are relatively inactive


The cII protein builds up much more quickly than cro
Therefore, the cII protein will turn on PRE


The l repressor is made
Thus, environmental conditions that are unfavorable
for growth promote the lysogenic cycle

This makes sense because there may not be sufficient
nutrients for the production of new bacteriophages
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14-74


After lysogeny is established, certain environmental
conditions can also favor induction to the lytic cycle
For example, exposure to UV light

recA (a cellular protein normally involved in DNA
recombination) detects the DNA damage




It cleaves the l repressor and inactivates it
This allows transcription from PR
Therefore, the cro protein will accumulate


It is activated to become a protease
Favoring the lytic cycle
This makes sense, because the exposure to UV light may
have already damaged the bacterium to the point where
further bacterial growth and division are prevented
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14-75
The OR Region Provides a Genetic
Switch Between the Two Cycles

To understand how this switch works, we need to
take a closer look at the OR region

The OR region contains three operator sites, designated
OR1, OR2, and OR3


These operator sites control two promoters, PR and PRM, which
transcribe in opposite directions
The l repressor protein or the cro protein can bind to any
or all of the three operator sites

This binding governs the switch between the lysogenic and the
lytic cycles
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14-76
The OR Region Provides a Genetic
Switch Between the Two Cycles


Two critical issues influence this binding

1. The relative affinities that the regulatory proteins have
for these operator sites

2. The concentrations of these regulatory proteins in the
cell
Refer to Figure 14.20
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14-77
Cro protein has the
highest affinity to OR3
and simiar affinity to
OR2 then OR1
l repressor has the
highest affinity to OR1
then OR2 then OR3
l repressor is a dimer
This binding inhibits
transcription from PR
So the lytic cycle is switched off
l repressor falls off
OR3 first
Figure 14.20
cro protein is a dimer
via
cooperative interaction
This binding blocks transcription from PRM
So the lysogenic cycle is switched off
PR is not
needed in
the later
stages of
the lytic
cycle
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
Genetic switches, like the one just described in
phage l, are also important in the developmental
pathways of bacteria and eukaryotes

For example

The choice between sporulation and vegetative growth in
bacteria

Initiation of cell differentiation during development in
eukaryotes
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14-79