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PowerPoint PresentationPoints for the lecture:Subunits have Distinct FunctionsLabeling the RNAP Active SiteActive Site is in the -SubunitSlide 6Slide 7RNAP ‘Footprint’ Changes Post-InitiationAbortive Initiation PredominatesPost-Initiation StepsSlide 11Slide 12Slide 13Slide 14Intrinsic Termination Depends on Adenine RunSlide 16Slide 17Initiation or Termination can be Used for RegulationSlide 19Slide 20Slide 21Prokaryotic Gene RegulationNon-Identical Sigma Factors Expand Regulatory CapacitySpecificity and Affinity are Driven by Multiple Cooperative InteractionsSlide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35What and Where are Operators?Promoter ‘Bashing’Linker-scanner Mutagenesis to Refine Position of Promoter Proximal ElementsSlide 39OperonSlide 41Slide 42Slide 43Biological Purpose of Lac OperonSlide 45Genetic Screen Based on Enzymatic ActivityNatural & Synthetic Small MoleculesSlide 48Studying the Lac OperonComplementation Analysis and Cis-Trans TestsSlide 51Slide 52Slide 53Slide 54Slide 55Slide 56Slide 57Allosteric Induced Conformational ChangesSlide 59Slide 60Slide 61Effects of Regulators on RNAP:Promoter StabilityEffect of Repressor on RNAPRepressor Dimers Bind on Same Face of DNARepressor Binds as a TetramerSlide 66Slide 67Slide 68Promoter Contains 3 Operator SitesHow to Resolve if Operator 2 or 3 is preferred?Chromatin Immunoprecipitation Assay is used to determine the relative DNA occupancy of a target proteinChIP-Seq can be used to determine DNA binding sites of a target proteinSlide 73Multiple RegulatorsSlide 75Transcriptional Response to Sugar LevelsLac Operon is Under + and - RegulationSlide 78Slide 79Slide 80Slide 81Slide 82Slide 83Slide 84Poor -10 & -35 Sequences at Lac PromoterSlide 86CAP (Catabolite Activator Protein) is a Positive Regulator (a.k.a. an Activator)Slide 88CAP Binds a Consensus Inverted Repeat Response ElementSlide 90CAP Binding Induces DNA BendingDetecting DNA BendingSlide 93Does Bent DNA Enhances RNAP Binding?Effect of CAP on RNAP:Promoter InteractionsSlide 963 Classes of CAP Regulated PromotersCAP Operators Display PeriodicityIn vitro Transcription AssayCAP:RNAP Contact Residues Can Be Genetically Identified at a Class I PromoterIn vivo and In vitro Activity is Similar for MutantsRNAP Contact Region Can Be Isolated by Second-Site Suppressor ScreenCAP Interacts with RNAP a-CTD at Class I PromotersGenetically Identified Contact Sites Are Physically the Contact SitesOrientation of CAP Operators is ImportantCAP has 2 RNAP Interaction Surfaces at Class II PromotersActivating Region (AR) is Upstream at Class II PromotersAR1 and AR2 Affect Different Steps in InitiationAR2 Surface Interacts with a-NTDDifferent Promoter Classes Utilize Different Protein RegionsSubunit Use Varies According to Class TypeCAP Can Function as a RepressorCombinatorial RegulationAdditional Bacterial RegulatorsSlide 115araBAD OperonSlide 117Slide 118Slide 119DNA Bending Requires both araO2 and araI SitesSlide 121Slide 122Slide 123Slide 1241Readings for next week:Book:Molecular BiologyChapters 7.2 (Lecture 5) & 8.3 (Lecture 6)Reviews:Transcription activation by catabolite activator protein (CAP). Busby, S. and Ebright, R.H.J Mol Biol. 1999 Oct 22;293(2):199-213.Bacterial nucleoid-associated proteins, nucleoid structure and gene expression.Dillon, S.C. and Dorman C.J.Nat Rev Microbiol. 2010 Mar;8(3):185-95.Journal Club Paper:Chromosome organization by a nucleoid-associated protein in live bacteria.Wang W, Li GW, Chen C, Xie XS, Zhuang X.Science. 2011 Sep 9;333(6048):1445-9.2Points for the lecture:•Transcription can be regulated •Bacteria (and viruses) optimized regulation by expressing multiple proteins in a single transcript (operon)•Small molecules can control gene programs•Protein:DNA contacts are driven by specific DNA sequences3Subunits have Distinct Functions4Labeling the RNAP Active Site5Active Site is in the -Subunit6SUBUNIT IS GREY, SUBUNIT IS CYAN, ’ SUBUNIT IS PINK AND SUBUNIT IS ORANGE-10 AND -35 REGIONS ARE IN YELLOWTransition from Closed (RPc) to Open Complex (Rpo)7DNA:RNAP Interactions8RNAP ‘Footprint’ ChangesPost-Initiation9Abortive Initiation Predominates10Post-Initiation Steps11Transcription Termination12Transcription Termination• Only certain regions of a genome are transcribed• Transcription complexes assemble at promoters and disassemble at the 3’ end of genes at specific termination sequences• Two types of termination sequences:(1) Rho-independent termination(2) Rho-dependent termination13Formation of an RNA Hairpin14Hairpin begins to formHairpin forms and destabilizes hybrid (RNA pull-out?)TerminationRho-Independent Termination15Intrinsic Termination Depends on Adenine Run16Rho-Dependent Termination17Rho-Dependent Termination of Transcription in E. coli18Initiation or Termination can be Used for Regulation19In log-phase E. coli–~4000 genes–~2000 core RNA polymerase molecules–~2/3 (1300) are active at a time–~1/3 (650) can bind σ subunits.–~1200 σ subunits.•Competition of σ for core determines much of a cell’s protein content.20Housekeeping vs. Regulated• Expression of housekeeping genes is constitutive (essentially)• Housekeeping genes have strong promoters and are efficiently and nearly continuously transcribed*housekeeping genes whose products are required at low levels have weaker promoters• Regulated genes are expressed at different levels under different conditions using specific operators/response elements within each promoter21Sigma Factor Directed Transcription Programs22Prokaryotic Gene RegulationE. coli can choose between 7 sigma factors and about 350 transcription factors to fine tune its transcriptional output An Rev Micro Vol. 57: 441-466 T. M. GruberSigma subunit Type of gene controlled # of genes controlledRpoD Growth/housekeeping ~1000RpoN N2; stress response ~15RpoS Stationary phase, virulence ~100RpoH Heat shock ~40RpoF Flagella-chemotaxis ~40RpoE ? ~5FecI Ferric citrate transport ~523Non-Identical Sigma Factors Expand Regulatory Capacity24Specificity and Affinity are Driven by Multiple Cooperative Interactions25Directing Simple and Cascade Responses26Sigma Factor Regulation2732 Controls Expression of the Proteins that Control It (a.k.a. Feedback loop)2832 Is Continuously Produced but Rapidly DegradedInvolved in cascade responses2932 Regulation is a Balanced Sensor30HSPs Reactivate 70 During
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