Transcript Chapter 17

Chapter 17
Regulation of Gene
Expression in Bacteria and
Bacteriophages
Copyright © 2010 Pearson Education Inc.
Chapter 19:
Regulation of Gene Expression in
Bacteria and Bacteriophages
Through evolutionary processes,
organisms have developed ways
to compensate for
environmental changes.
Alter gene activity to optimize
growth and reproduction in a
given environment.
Two Types of Genes
1) Regulated Genes – activity is controlled in response to the
needs of a cell or organism.
2) Constitutive genes - (housekeeping genes) always active
(e.g. protein synthesis and Glucose metabolism)
Basic Mechanism of Gene Regulations in Bacteria
Bacteria have developed ways to turn off genes whose products are
not needed and for turning on genes whose products are
needed in each environment.
The turning of genes off or on requires interaction between
regulatory proteins and DNA sequences.
Inducible Gene Expression
When a gene is turned on by the addition of a substance, it is called inducible gene.
The regulator substance is called an inducer, which are members of a class
of small molecules = effectors
Controlling site is near protein coding sequence.
The addition of inducer leads to induction.
Induction of genes required for lactose utilization in E. coli
E. coli grow in simple media containing salts, nitrogen sources, and glucose.
If lactose (or other sugar) is added instead of glucose,
a number of enzymes are rapidly synthesized.
*Inducer of Lactose operon
When Lactose is only sugar, three enzymes are synthesized
1) B-galactosidase
2) Lactose permease
3) Trans acetylase (function poorly understood)
All 3 genes are clustered on the genome and are transcribed
onto a single mRNA called a polygenic mRNA or a polycistronic mRNA
Chain terminating mutation
Nonsense mutants (chain terminating mutant) were used to determine that all 3 genes
were on the same mRNA.
Jacob & Monod’s Operon Model for
the regulation of the lac genes
• Operon is a cluster of genes, the expression of
which are regulated together by operator regulator
protein interactions, plus the operator region itself
and the promoter.
In E. coli, the lac operon is under
negative, positive and inducible control
Lac I+ gene encodes lac repressor protein made constitutively,
which will bind operator region of the lac operon.
Few repressors are present in cell since promoter is relatively weak.
Absence of lactose
Lac operon is under negative control:
There is a low level of lac gene expression because the repressor binds
and unbinds allowing for low amounts of protein such as
B-galactosidose and permease to be generated
Presence of lactose
Inducible Control
If in the presence of lactose, the above B-galactosidose produces
inducer molecules, allolactose, which is the inducer.
Experiments by Jacob and Monod
Partial diploid that has F‘ plasmid
Without inducer
With inducer
Mutation in Lac I gene, which generates a mutant repressor
that cannot bind to the operator
Without
inducer
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Mutation in Lac I gene, which generates a mutant repressor
that cannot bind to the operator
With inducer
Lac Operon experiments
Dominant Effect
Mutation in Lac I that
cannot bind Inducer but can bind
the operator
Lac Repressor model for tetramer protein structure
Has four polypeptides and
each polypeptide is from
repressor gene.
**This data convinced many scientist (at the time) that all genes
were under negative control due to the binding of a repressor. **
Positive control of the lac operon.
Turns on expression of the lac operon.
Ensures that lac operon stays on when lactose is the sole carbon source,
but not in the presence of glucose.
Glucose is used preferentially over lactose.
Positive control of the lac operon.
Glucose inactivates adenylate cyclase
Glucose removes remaining cAMP by
Activating Phosphodiesterase
In presence of glucose, concentration of
positive regulator (CAP-cAMP complex)
that binds the lac operon for increased gene
activity is reduced.
Because the presence of glucose reduces
the amount of cAMP in cell.
Basepair sequence of the lac I gene promoter
Shine-Dalgarno sequence
GUG start site
Basepair sequence of the controlling sites, promoter and operator, for the lac operon.
Tryptophan Operon
All necessary amino acids may not be present in a
growth medium. If a specific amino acid is
missing, the bacteria has certain operons that
enable the bacterial cell to manufacture that amino
acid.
For example the Tryptophan Operon
Has five structural genes
Two mechanisms of regulations for tryptophan operon
# 1 Repressor/operator interaction with tryptophan as the effector
molecule: Tryptophan binds the aporepressor (trpR) and then
binds operator to turn off the gene
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Two mechanisms of regulations for tryptophan operon
# 2 Attenuation Control Regulatory Leader region
Determines if initiated transcripts include other
structural genes or not.
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Operon called repressible operon (# 1). No transcripts in presence
of tryptophan.
The biosynthetic pathway is catalyzed by a specific enzyme
(at each step) which is coded by a specific gene or genes.
Presence of tryptophan in medium keeps operon turned off
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# 2 Attenuation Control
Absence of tryptophan or in the presence of low amounts of tryptophan:
Under severe tryptophan starvation
gene activity at maximum
 all long transcripts
Under less severe situation gene
Expression at less than maximum
Long and short transcripts
Greater the amount of tryptophan the greater the number of short transcripts
Attenuation controls = > terminates transcription producing short transcripts
mRNA
RNA polymerase response to
these mRNA secondary structures
Transcription and translation are tightly coupled in prokaryotes
Attenuation occurs at the mRNA level and can reduce transcription of trp-operon 8-or-10 fold.
*Last long enough for
Ribosome to load onto mRNA.
Position of ribosome on leader transcript determines if transcription is
terminated or not.
If starved for trytophan = lack trp-tRNA
If no trp-tRNA, ribosome stalls at trp codons. With Ribosome
on region one, the 1-2 loop can’t form.
So, the 2-3 loop for antitermination forms. Thus region 3 can’t
pair with region 4 and the RNA polymerase can now continues.
Starved for tryptophan
Transcription continues
++ trptophan
Termination signal
For RNA polymerase,
Which stops transcription
Attenuated-controlled bacterial operons
Regulation of other amino acid biosynthesis operons
Leader Peptides of other attenuated-controlled Bacterial operons
Isoleucine and leucine
The ara Operon of E. coli: Positive and Negative Control
At the same time that Jacob &
Monod were doing their work,
Englesberg, et. al. were studying
The regulation of the arabinose
(ara) operon of E. coli.
They found that instead of being
regulated with a negative control
mechanism as seen in the lac operon,
the ara operon was primarily under
Positive control. Although their
conclusion were not widely accepted,
biochemical and molecular test proved
that they were correct.
In the lac operon, allolactose would bind the repressor to remove it from the operator so that the
Polymerase could bind the operator and start transcription.
In the ara operon, two molecules of AraC protein bind and act as a bridge from the operator (araO 2 )
And to the promoter region ara I1 which creates a loop that prevents the binding of CAP-cAMP.
With the addition of arabionose, the arabionose bound AraC protein is allosterically modified to bind to
ara I2, which allows CAP-cAMP to bind the CAP site and positive regulate gene expression occurs.
However, for the ara operon to function, glucose can not be
Present. If present, it will eliminate cAMP and focus on the
Utilization of glucose
**Positive regulation of activators is now known to occur in a
Variety of prokaryotic systems and in all eukaryotes.
Bacteriophage Gene regulation
Regulation of gene expression in the lytic cycle and lysogeny
in baceriophage lambda (λ)
Excellent model for developmental switches in eukaryotic systems
After λ infection of bacteria, a choice is made between lytic and lysogenic pathways
1)
2)
Linear chromosome is circularized
in host
Transcription begins at PL & PR
PL promoter for left early operon
PR promoter for right early operon
These promoters are on different DNA
Strands.
Depends on a genetic switch, which involves competition between
the products of the CI gene (the repressor) and the Cro gene (the Cro protein)
regulator of CI gene.
Left
Right
Important info
cI gene
Cro gene
N gene
N = protein is the antiterminator
that allows RNA transcription
past transcription terminator signals.
Lysogeny
Lytic
cI protein
Integration of λ
cII protein stimulates
synthesis of cI repressor
which competes with
Cro protein.
Decision on which pathway
is taken is determined by the
amount of λ repressor or
Cro protein that is bound
to PR or OR region.
Overheads 1, 2 and 3