Transcript Brooker Chapter 14

CHAPTER 14
GENE REGULATION IN BACTERIA
AND BACTERIOPHAGES
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Introduction

The term gene regulation means that the level of
gene expression can vary under different conditions

Genes that have constant levels of expression are
termed constitutive
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sometimes called “housekeeping genes”
The benefit of regulating genes is that encoded
proteins will be produced only when required
Transcriptional Regulation

Most regulation of gene expression is at
transcriptional level

rate of RNA synthesis increased or decreased

Transcriptional regulation involves actions of two
types of regulatory proteins
 Repressors  Bind to DNA & inhibit transcription
 Activators  Bind to DNA & increase transcription

Negative control refers to transcriptional regulation
by repressor proteins
Positive control to regulation by activator proteins

Transcriptional Regulation

Small effector molecules affect transcription regulation

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bind to regulatory proteins not to DNA directly
effector molecule may increase transcription

inducers
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Bind activators & cause activator to bind DNA
Bind repressors & prevent repressor from binding DNA
Genes regulated this way are inducible
effector molecule may inhibit transcription

Corepressors
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Inhibitors
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bind repressors & cause repressor to bind DNA
bind activators & prevent activator from binding DNA
Genes regulated this way are repressible

Regulatory proteins have
two binding sites


One for a small effector
molecule
The other for DNA
gene regulation gods
Jacques Monod – Paris 1961
François Jacob & André Lwoff – 1953 CSH Symposium
Diauxic Growth Curve Demonstrated
Adaptation to Lac Metabolism
The lac Operon
Figure 14.3
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14-13
Regulatory Sequences of the Lac Operon
The Lac Operon Is Regulated both
Positively & Negatively

Negative - repressor protein - LacI
Positive - activator protein – CAP or CRP

Induction of Lac operon requires 2 events

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Release of repression

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Activation

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lactose binds to the lac repressor causing the repressor to release
operator site in DNA
cAMP binds CAP protein, cAMP-CAP dimerizes & binds CAP site
in DNA
Insures that operon is on only if


lactose is present
glucose is low
RNA pol
cannot initiate
transcription
Constitutive
expression
The lac operon is now
repressed
Figure 14.4
14-15
Lac repressor protein (violet)
forms a tetramer which binds to
two operator sites (red) located
93 bp apart in the DNA causing
a loop to form in the DNA. As a
result expression of the lac
operon is turned off. This model
also shows the CAP protein
(dark blue) binding to the CAP
site in the promoter (dark blue
DNA). The -10 & -35 sequences
of the promoter are indicated in
green.
Translation
The lac operon is now
induced
The conformation of the
repressor is now altered
Repressor can no longer
bind to operator
Figure 14.4
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14-16
Repressor does not completely
inhibit transcription
small amounts of the enzymes
are made
Figure 14.5
The cycle of lac operon induction & repression
The lacI Gene Encodes a Repressor
Protein

1950s, Jacob & Monod, & Arthur Pardee, identified
mutant bacteria with abnormal lactose adaptation

defect in lacI gene
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designated lacI– I = induction mutant
caused constitutive expression of lac operon
(ie in absence of lactose)
The lacI– mutations mapped very close to the lac operon
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Jacob, Monod & Pardee hypothesized 2 ways for
lacI to function
This hypothesis predicts that
lacI works in trans manner
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This hypothesis predicts that
lacI works in a cis manner
Used genetic approach to test hypotheses
PaJaMo Experiment
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Used F’ plasmids carrying part of lac operon
Put into mutant bacteria by conjugation
Bacteria that get F’ have 2 copies of lacI
gene
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merodipoloids
PaJaMo Experiment
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2 lacI genes in a merodiploid are alleles
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lacI– on the chromosome
lacI+ on the F’ factor
Genes on F’ plasmid are trans to bacterial
chromosome
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If hypothesis 1 is correct
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repressor produced from F’ plasmid can regulate the lac
operon on the bacterial chromosome
If hypothesis 2 is correct

binding site on F’ plasmid cannot affect lac operon on the
bacterial chromosome, because they are not physically
adjacent
PaJoMo Experiment
Figure 14.7
14-23
Figure 14.7
14-24
Figure 14.7
14-25
Results
Lactose addition has no effect
because operon is already on
Induction is restored in merodiploid.
Now lactose addition is required to
turn operon on
Wildtype
Induction
mutants
From Jacob & Monod, 1961, J Mol Biol 3:318
Analysis of Lac Operon Mutants
-
lacI
I+O+Z-Y+
F’I-O+Z+Y+
From Jacob & Monod, 1961, J Mol Biol 3:318
Analysis of Lac Operon Mutants
-
-
Mutation is
cis
• In merodiploid, LacZ constitutive, but LacY inducible
• OC only controls transcription of DNA on which OC is
located
• O (operator) is cis-regulatory element
Interpreting the Data
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The interaction between regulatory proteins & DNA
sequences have led to two definitions

Trans-effect & trans-acting factor
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Genetic regulation that can occur even though DNA segments are
not physically adjacent
Mediated by genes that encode DNA-binding regulatory proteins
Example: The action of the lac repressor on the lac operon
Cis-effect & cis-acting element
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A DNA sequence adjacent to the gene(s) it regulates
Mediated by sequences that are bound by regulatory proteins
Example: The lac operator
Genetic Implications of Trans vs Cis
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mutations in trans-acting factors complemented by
2nd wt gene
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mutations in cis-acting elements ARE NOT
complemented by 2nd wt element
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Trans interactions (complementation) indicate
mutation in structural gene
Cis interactions indicate mutations in regulatory
sequences
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Wildtype
Induction
suppression
mutant –
Dominant
Negative
From Jacob & Monod, 1961, J Mol Biol 3:318
Dominant Inhibitors or
Dominant Negatives
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Proteins with multiple functional domains &
form multimeric complexes may be altered
to prevent one function, but allow the other
When mutants retain ability to form
multimeric complexes, dominant inhibition
may occur
Analysis of Lac Operon Mutants
Mutation is
trans
Dominantnegative
Mutation disrupts ligand binding domain of repressor
Analysis of Lac Operon Mutants
Mutation disrupts DNA binding domain of repressor
lac Operon Also Regulated By Activator Protein

catabolite repression
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When exposed to both lactose & glucose
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E. coli uses glucose first, & catabolite repression
prevents the use of lactose
When glucose is depleted, catabolite repression is
alleviated, & the lac operon is expressed
The sequential use of two sugars by a bacterium is
termed diauxic growth
The lac Operon Is Also Regulated By an Activator
Protein

Effector molecule in catabolite repression cAMP
(cyclic AMP)

cAMP is produced from ATP by adenylyl cyclase
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cAMP binds activator protein CAP or CRP
(Catabolite Activator Protein) or (cyclic AMP receptor protein)
States of Lac Regulation
(b) Lactose but no cAMP
Figure 14.8
States of Lac Regulation
Figure 14.8
The trp Operon

The trp operon (pronounced “trip”) is involved in the
biosynthesis of the amino acid tryptophan
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The genes trpE, trpD, trpC, trpB & trpA encode enzymes
involved in tryptophan biosynthesis
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The genes trpR & trpL are involved in regulation

trpR  Encodes the trp repressor protein
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
Functions in repression
trpL  Encodes a short peptide called the Leader peptide

Functions in attenuation
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14-44
Figure 14.13 Organization of the trp operon & regulation via the trp
repressor protein
Med
Another mechanism
of regulation
Figure 14.13 Organization of the trp operon & regulation via the trp
repressor protein
14-47
RNA pol can bind
to the promoter
Cannot bind to
the operator site
Figure 14.13 Organization of the trp operon & regulation via the trp
repressor protein
14-45
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Attenuation occurs in bacteria because of the coupling of
transcription & translation
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During attenuation, transcription actually begins but it is
terminated before the entire mRNA is made
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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
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The first gene in the trp operon is trpL
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It encodes a short peptide termed the Leader peptide
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Region 2 is complementary to regions 1 & 3
Region 3 is complementary to regions 2 & 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

Therefore, the formation of the 3-4 stem-loop
causes RNA pol to terminate transcription at the
end of the trpL gene
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Conditions that favor the formation of the 3-4
stem-loop rely on the translation of the trpL mRNA
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There are three possible scenarios
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1. High levels of tryptophan
2. Medium levels of tryptophan – high trp-tRNA
3. Low levels of tryptophan – med-low trp-tRNA
Repression occurs
Figure 14.13 Organization of the trp operon & regulation via the trp
repressor protein
Attenuation occurs
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
Med
Figure 14.15 Possible stem-loop structures formed from trpL mRNA under
different conditions of translation
Transcription occurs
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
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

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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