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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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Figure 14.1
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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
Negative control: Repressors  Bind to DNA
and inhibit transcription
Positive control: Activators  Bind to DNA and
increase transcription
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14-5

In some cases, the presence of a small effector molecule
may increase transcription
 These molecules are termed inducers
 Bind activators and cause them to bind to DNA
 Bind repressors and prevent them from binding to DNA

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
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14-6
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Promoter
Fig. 14.2ab(TE Art)
DNA
Repressor
protein
No transcription
RNA
polymerase
or
Transcription
occurs
Inducer
Repressor
protein
Repressor protein, inducer molecule, inducible gene
RNA
polymerase
Transcription
occurs
Repressor protein
or
Corepressor
Repressor protein
No transcription
Repressor protein, corepressor molecule, repressible gene
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transcription or Art)
Fig.No14.2cd(TE
Activator
protein
Inducer
RNA
polymerase
Activator
protein
Transcription
occurs
Activator protein, inducer molecule, inducible gene
RNA
polymerase
Activator
protein
Transcription or
occurs
No transcription
Inhibitor
Activator
protein
Activator protein, inhibitor molecule, repressible gene
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
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14-10
Figure 16-3
Copyright © 2006 Pearson Prentice Hall, Inc.

An operon contains several different regions

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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Figure 14.3
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14-13


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
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14-12
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
Figure 16-5
Copyright © 2006 Pearson Prentice Hall, Inc.
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
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Figure 14.7
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Figure 14.7
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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
Figure 16-6
Copyright © 2006 Pearson Prentice Hall, Inc.
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14-30
Figure 16-7
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Table 16-1
Copyright © 2006 Pearson Prentice Hall, Inc.
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
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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
Figure 16-9
Copyright © 2006 Pearson Prentice Hall, Inc.
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
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14-33
Figure 18.23 Positive control of the lac operon by
catabolite activator protein (CAP)
Promoter
DNA
lacl
lacZ
CAP-binding site
Active
CAP
cAMP
Inactive
CAP
RNA
polymerase
can bind
and transcribe
Operator
Inactive lac
repressor
(a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized.
If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces
large amounts of mRNA for the lactose pathway.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Promoter
DNA
lacl
lacZ
CAP-binding site
Operator
RNA
polymerase
can’t bind
Inactive
CAP
Inactive lac
repressor
(b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized.
When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Both
negative and
positive
control
mechanisms
are used by
the cell.
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



But not O2 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

This facilitates the binding of the repressor protein
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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
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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
Figure 16-15
Copyright © 2006 Pearson Prentice Hall, Inc.
Regulation of a anabolic pathway
(a) Regulation of enzyme
activity
Precursor
Feedback
inhibition
Enzyme 1
Enzyme 2
Enzyme 3
(b) Regulation of enzyme
production
Gene 1
Gene 2
Regulation
of gene
expression
Gene 3
–
Enzyme 4
Gene 4
–
Enzyme 5
Tryptophan
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Gene 5
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
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

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