Final ExamPowerPoint PresentationSlide 3Cycling Through Complexes Within MinutesGreen Fluorescent Protein (GFP)GFP Derivatives and Other Fluorescent ProteinsFusion Proteins Often Used for Localization StudiesVisualizing GR:GRE Complexes In vivoHormone-Dependent Localization of GFP-GR to the MMTV LocusLocalizing MMTV DNA Locus by FISHFISH AnalysisGFP-GR Colocalizes with Nascent MMTV TranscriptsFRAP, FLIP & PhotoactivationMobility Assessment by Fluorescence Recovery After Photobleaching (FRAP)Measuring Dynamics is Accomplished Using Live CellsSlide 16GR Dynamically Associates with GREsSlide 18Promoter Associated GRIP1 is DynamicDynamics Allows Rapid Sequential RecruitmentSlide 21RNAP II Exchange is Relatively SlowNon-engaged RNAP II is DynamicTranscription Complexes Bind DNA TransientlyWhat Drives the Dynamics?Molecular Chaperones and Proteasome Components Interact LateProteasome Inhibitors Slow GR CyclingHormone-Dependent Molecular Chaperone ColocalizationIn vitro Molecular Chaperone AnalysisFluorescence Anisotropy DNA Binding Assayp23 Selectively Disassembles Receptor RE ComplexesHormone and DNA Alter p23-Receptor Interactionsp23 Can Disassemble Receptor:RE ComplexesIn vivo, IRs Bind DNA in Association with CofactorsLXXLL Peptide Can Reestablish TR:TRE ComplexesLXXLL and p23 Effects Are in EquilibriumSlide 37p23 and GRIP1 Compete for GR Association In vivoInfluence on chromatin status?Slide 40Slide 41Slide 42Do dynamics have a physiological use?Serum Hormone Levels FluctuateSlide 45So…Slide 47Slide 48Slide 49Slide 50Protein Dynamics Permit Transitions between Structures and PTMs Guide the PathwayThank you & Good Luck!Final ExamTest on December 8th 8:30-11:30 a.m.Open book/Open NoteExample exams are on-line in the MCB502 Folder2Points for the lecture:Transcription complexes are dynamicComplexes are actively turned overDynamics are critical to progress multi-component systemsPost-Translation Modification (PTM) guide pathways3DNA-Bound Complexes are Inherently Stable…assembly is spontaneous, but depends on cell and response element contexts4Cycling Through Complexes Within Minutes-Ac-Ac-AcMediatorMediator Recruitment-Ac-Ac-AcMediatorRNAPII RecruitmentChIP Results5Green Fluorescent Protein (GFP)Aequorea victoria6GFP Derivatives and Other Fluorescent Proteins7Fusion Proteins Often Used for Localization Studies8Visualizing GR:GRE Complexes In vivoIntegrated (MMTV LTR)200; GR-GFP:9Hormone-Dependent Localization of GFP-GR to the MMTV Locus(-) Dexamethasone (+) Dexamethasone10Localizing MMTV DNA Locus by FISH11FISH Analysis12GFP-GR Colocalizes with Nascent MMTV TranscriptsGFP-GR MMTV RNA FISH Image Overlay13FRAP, FLIP & Photoactivation14Mobility Assessment by Fluorescence Recovery After Photobleaching (FRAP)15Measuring Dynamics is Accomplished Using Live CellsFixedLiveSeconds After Photobleaching0010.62.919.75.430.47.916GR Dynamics Visualized by FRAP17GR Dynamically Associates with GREsMobile Fractionnon18DNA Bound GR is Dynamic…but what about the coactivators19Promoter Associated GRIP1 is Dynamic20Dynamics Allows Rapid Sequential Recruitment21…and RNA Polymerase II?22RNAP II Exchange is Relatively Slow23Non-engaged RNAP II is Dynamic24Transcription Complexes Bind DNA TransientlyAporeceptorComplexHormoneHoloreceptor:RE ComplexRegulatory/InitiationComplexes25What Drives the Dynamics?26Molecular Chaperones and Proteasome Components Interact Late27Proteasome Inhibitors Slow GR Cycling28Hormone-Dependent Molecular Chaperone Colocalization29In vitro Molecular Chaperone Analysis30Fluorescence Anisotropy DNA Binding Assay31p23 Selectively Disassembles Receptor RE Complexes Anisotropy value32Hormone and DNA Alter p23-Receptor Interactions33p23 Can Disassemble Receptor:RE Complexes34In vivo, IRs Bind DNA in Association with CofactorsCoregulatorsHistone Acetyl Transferase p300/CBP p160s (GRIP1, SRC1)Chromatin Remodeling BAF250Mediator TRAP220/DRIP20535LXXLL Peptide Can Reestablish TR:TRE ComplexesAnisotropy valuefluorescence anisotropy:36LXXLL and p23 Effects Are in EquilibriumAnisotropy valuefluorescence anisotropy:37Balancing Assembly and Disassembly38p23 and GRIP1 Compete for GR Association In vivoDsRed-p23 GFP-GR++----2.5+/-0.1DsRed-p23+++---3.2+/-0.1GRIP1+--+--2.7+/-0.1DsRed-GRIP1+--++-3.3+/-0.1p23+--+-+2.6+/-0.1p2335+-----3.3+/-0.1GFP-GRLifetime (nanoseconds)39Influence on chromatin status?Mapping Global DNase I Hypersensitive Sites (Open Chromatin)Transcription Factor DNA Occupancy Increases in p23 Dp23 is Required for Proper DHS Pattern32608691518(1570)p23specificOverlapWT specificTotal WT DNase I Hypersensitive Sitesp23DHSs43Do dynamics have a physiological use?44Serum Hormone Levels FluctuateSerum cortisol (mg/100 mL)Time of Day810121416181020045Continuous Disassembly Enables Signal MonitoringAssemblySignalingDisassemblyATPATPATP46So…What did we learn?47Dynamics is a General Feature for Cellular FactorsDynamic rearrangement of surface proteins is essential for cytokinesisRapid transport of proteins and protein cargoChemotaxis and Cell MotilityProtein translationPathways occur in context-dependent set orders Despite the potential for Chaos48Self-Assembly vs. Self-Organization49Transitioning Through Multi-Step PathwaysHAT Regulatory ComplexChromatin RemodelingPreinitiation Complex50SGRGKGGKGLGKGGAKRHRC AcSGRGKGGKGLGKGGAKRHRC Ac AcSGRGKGGKGLGKGGAKRHRCAc AcKPGQPMYKGKKALRRQETVDALTAF250~KD (M)>250405>1000Post-translation Modifications (PTMs) Guide Pathways51Protein Dynamics Permit Transitions between Structures and PTMs Guide the PathwayHAT action leads to chromatin remodelingHistone removal& PTN addition leads toMediator nucleation Coregulatory assembly promotes PIC formationRNAP IIreleasesActivated transcription factor initiates gene response by binding to its cognate response element52Thank you& Good
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