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Unformatted text preview: Transcription Regulation:
1. Control of transcription initiation in response
to a change in the environment.
2. Sigma Factors
3. Anti-sigma factors Stress
• Stress in an abiotic factor or set of
factors that limits the production of
• Examples of stress factors are
unfavorable temperature, pH, nutrient
availability, water availability, oxygen
concentration, etc. Sense and Adapt to Stress
• E. coli grows optimally at 37°C and dies at
• What happens to E. coli at 42°C – a stress
• Is E. coli sensing this non-optimal
temperature and responding to limit the
damages caused by the high temperature?
• If so, then E. coli grown at 42° should be
better able to deal with the effects of 55° than
E. coli grown at 37°. How is E. coli able to sense and
adapt to the stress of 42°?
• Ask what changes about cells grown at 37°
versus 42° - specifically what proteins
• Cells were treated with 35S-methionine for 1
min to label the most highly expressed
proteins in the cell. These proteins were
analyzed by SDS-PAGE. The proteins were
visualized by exposing the SDSpolyacrylamide gel to film. DnaK and GroE proteins are synthesized at
a higher rate after temperature upshift DnaK
GroE DnaK and GroEL are chaperones that help to fold proteins.
From: Grossman AD et al. 1984 Cell 82:383-390 How doe the levels of DnaK and
GroE increase in 42° grown cells?
gene transcription translation protein
degraded Ask if levels of mRNA change? Northern Blot Levels of GroE mRNA increase after
temperature upshift WT From: Yamamori T. and Yura T. 1982 PNAS 79:860-864 What is increasing the levels of
groE mRNA in cells grown at 42°?
There is a protein that increases transcription of groE and
this protein is only active or present in 42° cells.
If so, we should be able to isolate a mutant strain of E.
coli in which the gene encoding this protein is mutated
Such a mutant should have decreased expression of
groE at 42°. RpoH is required for increased
transcription of groE after shift to 42° WT rpoH From: Yamamori T. and Yura T. 1982 PNAS 79:860-864 RpoH – A sigma factor?
• RpoH protein shares amino acid sequence
similarity with sigma-70, the sigma subunit of
• The sigma subunit of RNA polymerase makes
contacts with the DNA and recognizes the
• Hypothesis: RpoH is an alternative sigma factor
that directs RNA polymerase to a groE and other
heat-inducible promoters. In Vitro
• DNA containing test
promoter - groE
promoter (PHS) - and
control promoter – galK
• Incubate DNA with RNA
• Analyze radioactive
RNA products by
polyacrylamide gel. RpoH (Sigma-32) directs transcription of
Core RNA polymerase From: Grossman AD et al. 1984 Cell 82:383-390 Types of Sigma Factors
• House-keeping – required for growth
• Alternative sigma factors – not required
for growth under most environmental
conditions RpoH is required for transcription of
groE, but why is groE only
transcribed at 42°?
• Higher levels of RpoH is 42° cells.
• RpoH is an unstable protein.
• At 32°, RpoH is degraded by proteases and
• At 42°, the proteases and chaperones work on
proteins unfolded by the heat. These levels of
RpoH increase and activate expression of genes
for more production of proteases and
chaperones. Sigma-28(FilA) activates expression of late
flagellar genes only after assembly of the
hook and basal body of the flagellum Hughes KT et al. 1993 Science 262:1277-1280 Hypothesis
• There is a protein that inhibits sigma-28 (FliA) activity
until the basal body-hook are assembled.
• If so, then should be able to isolate a mutant strain in
which the gene encoding this inhibitory protein is
mutated and non-functional.
• Such a mutant should have increased expression of
late-flagellar gene (fljB-flagellin) in a mutant strain
background that is unable to assemble the basal
body-hook (fla-). Loss of the FlgM protein allows expression
of the flagellin gene in a strain lacking
lacking a gene for the basal body Hughes KT et al. 1993 Science 262:1277-1280 Does FlgM protein act directly to inhibit
transcription via RNA polymerase
containing sigma-28 (FliA)?
• In vitro transcription assay
• DNA containing test promoter - fliC promoter
- and control promoter – Ptac promoter
• Incubate DNA with RNA polymerase and
• Analyze radioactive RNA products by
polyacrylamide gel. Purified FlgM inhibits transcription by RNA
polymerase with sigma-28 What relieves FlgM inhibition of sigma-28 when the
basal body-hook is assembled?
Ohnishi K. et al. 1992 Mol. Microbiol. 6:3149-3157 Hughes KT and Mathee K 1998 Annu. Rev. Microbiol. 52:231-86 ...
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This note was uploaded on 11/23/2009 for the course MIMG 101 taught by Professor Bradley during the Winter '08 term at UCLA.
- Winter '08