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mol14 - Chapter 6!end Key terms and questions except 7 8 15...

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The Big Chapter 7 OH this week Mon and Wed (2-3pm) No Tuesday OH Chapter 6 end - Key terms and questions except 7, 8, 15 Today’s Experiments and Techniques – biochemical definition of 3 distinct RNA polymerases – defining important sequences for transcription consensus sequences 5’ deletion/mutagenesis series S1 nuclease protection – detecting protein:DNA binding DNaseI footprinting gel-shift (EMSA) assays
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Transcriptional regulation Overview The polymerases Regulatory sequences finding them building complexity Regulators finding them ʻ dissecting ʻ them Bacterial principles … more than one
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RNA polymerase does not act alone: σ (sigma) factors help ʻ find ʼ the promoter MCB 4-12 Core polymerase ( α , α , β , β ʼ , ω ) Plus σ = holoenzyme -50 -35 -10 +1 +20 σ 70 promoter Many different σ factors: Table 7-1 ʻ alternative σ factors ʼ
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Transcription can be activated or repressed: a more sophisticated view of the lac operon Multiple regulators, multiple regulatory sites OFF, repressed ON; not activated, not repressed ON, activated
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Regulation by distant sequences: enhancers and activators Low [gln] NtrB phosphorylates NtrC Looping, stimulation of bound (inactive) σ 54 -Pol Two-component regulatory system: NtrB - sensor NtrC- response regulator Other examples:PhoR/PhoB, His/Asp P-relay, HOG
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MCB 7-8 three distinct eukaryotic RNA polymerases Experiment: Make protein extracts Separate by ion-exchange chromatography (p. 96) Assay fractions for transcriptional activity Total protein RNA synthesized
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Chromatography MCB:3-37
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MCB 7-8 three distinct eukaryotic RNA polymerases Experiment: Make protein extracts
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