Download Control of Gene Expression in Bacteria

Control of Gene
Expression in Bacteria
17
BIOLOGICAL SCIENCE
FOURTH EDITION
SCOTT FREEMAN
Lectures by Stephanie Scher Pandolfi
© 2011 Pearson Education, Inc.
Key Concepts
In bacteria, gene expression can be controlled at three levels:
transcription, translation, or post-translation (protein activation).
Changes in gene expression allow bacterial cells to respond to
environmental changes.
Transcriptional control can be negative or positive. Negative
control occurs when a regulatory protein prevents transcription.
Positive control occurs when a regulatory protein increases the
transcription rate.
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Introduction
• A cell does not express all of its genes all of the time. Instead, they
are very selective about the genes they express, how strongly they
are expressed, and when they are expressed.
• Gene expression occurs when a gene product is actively being
synthesized and used in a cell. Regulation of gene expression is
critical to the efficient use of resources and thus survival.
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Gene Regulation and Information Flow
• Escherichia coli has served as an excellent model organism for the
study of prokaryotic gene regulation.
• Like most bacteria, E. coli can use a wide array of carbohydrates to
supply carbon and energy. Control of gene expression allows E.
coli to respond to its environment and switch its use of sugars.
• Gene expression in bacteria was predicted to be triggered by
specific signals from the environment.
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Mechanisms of Regulation―An Overview
• Information flow occurs in three steps, represented by arrows:
DNA
mRNA
protein
activated protein
• Genes can be under transcriptional, translational, or posttranslational control.
– All three types of regulation occur in bacteria.
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Transcriptional Control of Gene Expression
• Transcriptional control occurs when the cell does not produce
mRNA for specific enzymes.
– The cell avoids the production of these enzymes by utilizing
regulatory proteins that prevent RNA polymerase from binding
to a promoter.
DNA
x
mRNA
protein
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activated protein
Translational Control of Gene Expression
• Translational control allows the cell to prevent the translation of
an mRNA molecule that has already been transcribed. This can
occur through many mechanisms:
– Regulatory molecules can speed up mRNA degradation.
– Translation initiation can be altered.
– Translation proteins can be affected.
DNA
mRNA
x
protein
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activated protein
Post-Translational Control of Gene Expression
• Post-translational control occurs when the cell fails to activate a
manufactured protein by chemical modification.
DNA
mRNA
protein
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x
activated protein
Control of Gene Expression in Bacteria
All three forms of gene expression control occur in bacteria.
•
Transcriptional control is slow but efficient.
•
Translational control allows a cell to quickly change which
proteins are produced.
•
Post-translational control provides the most rapid response but is
energetically expensive.
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© 2011 Pearson Education, Inc.
Mechanisms of Regulation―An Overview
• The level of expression of different genes can be highly variable.
Variation in gene expression allows cells to respond to changes in
their environment.
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Metabolizing Lactose―A Model System
• E. coli’s preferred carbon source is glucose, and uses lactose only
when glucose is depleted.
• Before it can utilize lactose, E. coli must transport it into the cell,
where the enzyme b-galactosidase can cleave it to produce glucose
and galactose.
• E. coli produces high levels of b-galactosidase only when lactose is
present in the environment.
• Thus, lactose acts as an inducer—a molecule that stimulates the
expression of a specific gene.
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Identifying Genes under Regulatory Control
• To find the genes that code for b-galactosidase and the membrane
transport protein that brings lactose into the cell, Monod and
François Jacob isolated and analyzed E. coli mutants that could not
metabolize lactose.
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Finding Mutants for a Particular Trait
• Isolating mutants with respect to a particular trait is a two-step
process.
1. Generate a large number of individuals with mutations at
random locations in their genomes.
2. Use genetic screening on the mutants to find individuals with
defects in the process or pathway in question.
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Replica Plating to Find Mutant Genes
• Replica plating, a process used to identify mutant cells, is a fourstep process.
1. Grow bacterial colonies on master plates containing a
medium with many sugars.
2. Transfer cells from each colony to a piece of sterilized velvet.
3. Transfer cells to a plate with a medium containing only
lactose to screen for colonies that could not grow on lactose.
This new plate is called a replica plate.
4. Compare the colonies on the master plate and replica plate.
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Replica Plating to Find Mutant Genes
• Indicator plates can also be used.
– These allow researchers to directly observe mutants with
metabolic deficiencies.
– These plates made it possible for them to identify colonies
without functioning β-galactosidase.
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Different Classes of Lactose Metabolism Mutants
• The three genes involved in lactose metabolism were named lacZ,
lacY, and lacI.
• Three classes of E. coli mutants defective in lactose metabolism
were identified:
1. lacZmutants lack functional b-galactosidase.
2. lacY mutants lack the membrane protein galactoside
permease and so cannot transport lactose into the cell.
3. lacI mutants are called constitutive mutants because they
produce b-galactosidase and galactoside permease even when
lactose is absent.
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Several Genes Are Involved in Metabolizing Lactose
• The lacZ and lacY genes code for proteins involved in lactose
metabolism, while the lacI gene product serves a regulatory
function.
• In the absence of lactose, the lacI gene product shuts down
expression of lacZ and lacY. When lactose is present, however,
transcription of lacZ and lacY is induced.
• Further studies revealed that the lacZ, lacY, and lacI genes are
located close together on the circular E. coli chromosome. This
suggested that lacI could control both lacZ and lacY.
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Mechanisms of Negative Control: The Repressor
• Transcription can be regulated via negative control or positive
control.
Negative control occurs when a regulatory protein binds to DNA
and shuts down transcription.
Positive control occurs when a regulatory protein binds to DNA
and triggers transcription.
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Mechanisms of Negative Control: The Repressor
• Szilard and Monod hypothesized that the lacI gene codes for a
repressor that exerts negative control over the lacZ and lacY genes.
– They hypothesized that the repressor would bind directly to the
DNA on or near the promoter for the lacZ and lacY genes.
– Lactose then would interact with the repressor in a way that
makes the repressor release from its binding site.
– Lactose thus would induce transcription by removing negative
control.
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The lac Operon
• Jacob and Monod coined the term operon for a set of coordinately
regulated bacterial genes that are transcribed together into one
mRNA.
– The group of genes involved in lactose metabolism was thus
termed the lac operon.
• Later a fourth gene, called lacA, was discovered to be part of the lac
operon. The lacA gene codes for the enzyme transacetylase, which
has a protective function.
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The lac Operon
• Three hypotheses are central to the Jacob-Monod model of lac
operon regulation:
1. The lacZ, lacY, and lacA genes are transcribed together and
are thus coordinately regulated.
2. The lacI protein is a repressor that prevents transcription of
the lac operon by binding to a site called the operator.
3. Lactose, the inducer, binds directly to the lacI repressor,
causing it to release from the operator and ending negative
control of the operon.
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The lac Operon
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Why Has the lac Operon Model Been So Important?
• Regulation of the lac operon provided an important model system
in genetics.
• We now know that gene expression of many bacterial operons is
regulated by physical contact between regulatory proteins and
specific regulatory sites on DNA.
• In addition, as in the lac operon, the activity of many other key
regulatory proteins is regulated by post-translational control.
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Mechanisms of Positive Control: Catabolite Repression
• Transcription of the lac operon is greatly reduced when glucose is
present, even when lactose is also available.
– When glucose is already available, the cell does not need to
produce more by cleaving lactose.
• This is an example of catabolite repression.
– Occurs when one of the product molecules (the catabolite) of a
reaction represses the production of the enzyme(s) responsible
for that reaction.
– In the case of the lac operon, glucose is the catabolite.
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The CAP Protein and Binding Site
• The absence of glucose activates expression of the lac operon
through positive control.
• The catabolite activator protein (CAP) binds the CAP binding
site near the lac promoter and triggers transcription.
• CAP binding strengthens the lac promoter to increase expression.
• CAP is regulated by cyclic AMP (cAMP) binding to it. Only when
CAP is bound to cAMP can it bind DNA.
• If cAMP levels are low, CAP is not active and transcription is not
increased.
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Glucose Influences Formation of the CAP-cAMP Complex
• When extracellular glucose concentrations are high, intracellular
cAMP concentrations are low; when extracellular glucose
concentrations are low, intracellular cAMP concentrations are high.
• The enzyme adenylyl cyclase produces cAMP from ATP and is
inhibited by extracellular glucose.
• The amount of cAMP and the rate of lac transcription are inversely
related to the concentration of glucose.
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Summary of Control of the lac Operon
• Because positive and negative control elements are superimposed,
E. coli fully activates the genes for lactose metabolism only when
lactose is available and when glucose is scarce or absent.
© 2011 Pearson Education, Inc.
© 2011 Pearson Education, Inc.