Slide_Sigmas_2009

Slide_Sigmas_2009 - Transcription Regulation: Sigma Factors...

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Unformatted text preview: Transcription Regulation: Sigma Factors Outline 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 biomass. •  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 55°C. •  What happens to E. coli at 42°C – a stress condition? •  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 change? •  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 Expression 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°? Hypothesis: 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 and non-functional. 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 RNA polymerase. •  The sigma subunit of RNA polymerase makes contacts with the DNA and recognizes the promoter elements. •  Hypothesis: RpoH is an alternative sigma factor that directs RNA polymerase to a groE and other heat-inducible promoters. In Vitro Transcription assay • DNA containing test promoter - groE promoter (PHS) - and control promoter – galK promoter • Incubate DNA with RNA polymerase and nucleotides (32P-CTP) • 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 chaperones. •  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 nucleotides (32P-CTP) •  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.

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