分子生物学第四次课

TranscriptionalFactorandRNAProcessingWangDi（王迪）Ph.D2010-9-15InstituteofImmunology,[e-mail]ingEukaryoticGenesComputationallyüContent-basedMethodsGCcontent,hexamerrepeats,compositionstatistics,codonfrequenciesüSite-basedMethodsdonorsites,acceptorsites,promotersites,start/stopcodons,polyAsignals,lengthsüComparativeMethodssequencehomology,ESTsearchesüCombinedMethodsPromoterpredictionCpGislandCpGislandPredictionIdentificationoftranscriptionalstartsiteandcorepromoterSoftwareprediction:“Corepromoter”and“NeuralNetworkPromoterPrediction”02004006008001000120014001600180020000.00.20.40.60.81.0Scorebase02004006008001000120014001600180020000.00.20.40.60.81.0ScorebaseIdentificationoftranscriptionalstartsiteandcorepromoterIdentificationoftranscriptionalstartsiteandcorepromoterIdentificationoftranscriptionalstartsiteandcorepromoter5’-RACE:RapidAmplificationofcDNAEndsPrimerExtensionFunctionalanalysisofputativepromoterReportergenestrategyFunctionalanalysisofputativepromoterReportergenestrategySubcellularlocalizationanalysisusingGFP-recombinantproteinIdentificationofcis-transcriptionalelementDeletionanalysisofcorepromoterDeletionanalysisofcorepromoterPromoterDatabases:TRANSFACisadatabaseoneukaryoticcis-actingregulatoryDNAelementsandtrans-actingfactors.Itcoversthewholerangefromyeasttohuman.BiologicalDatabases/BiologischeDatenbankenGmbHInrelease4.0,itcontains8415entries,4504ofthemreferringtositeswithin1078eukaryoticgenes,thespeciesofwhichrangingfromyeasttohuman.Additionally,thistablecomprises3494artificialsequenceswhichresultedfrommutagenesisstudies,invitroselectionProceduresstartingfromrandomoligonucleotidemixturesorfromspecifictheoreticalconsiderations.Andfinally,thereare417entrieswithconsensusbindingsequencesgivenintheIUPACcode.MatInspectorSearchforpotentialtranscriptionfactorbindingsitesinyourownsequenceswiththematrixsearchprogramMatInspectorusingtheTRANSFAC4.0matrices.FastMAprogramforthegenerationofmodelsforregulatoryregionsinDNAsequences.FastMusingtheTRANSFAC3.4matrices.PatSearchSearchforpotentialtranscriptionfactorbindingsitesinyourownsequenceswiththepatternsearchprogramusingTRANSFAC3.5TRRD3.5sites.FunSitePRuninteractivelyFunSiteP.RecognitionandclassificationofeukaryoticpromotersbysearchingtranscriptionfactorbindingsitesusingacollectionofTranscriptionfactorconsensi.://•TheEukaryoticPromoterDatabaseisanannotatednon-redundantcollectionofeukaryoticPOLIIpromoters,forwhichthetranscriptionstartsitehasbeendeterminedexperimentally.•Theannotationpartofanentryincludesdescriptionoftheinitiationsitemappingdata,cross-referencestootherdatabases,andbibliographicreferences.•EPDisstructuredinawaythatfacilitatesdynamicextractionofbiologicallymeaningfulpromotersubsetsforcomparativesequenceanalysis.•EPDEXisacomplementarydatabasewhichallowsuserstoviewavailablegeneexpressiondataforhumanEPDpromoters.EPDEXisalsoaccessiblefromtheISREC-TRADATdatabaseentryserver.TESSTranscriptionElementSearchSystemComputationalBiologyandInformaticsLaboratory,SchoolofMedicine,UniversityofPennsylvania,1997://darwin.nmsu.edu/~molb470/fall2003/Projects/solorz/(PROMOTER2.0)DensityofTFfromEPD(PromoterScan)Searchesofweightmatricesagainstatestsequence(TFSearch/TESS)://bimas.dcrt.nih.gov/molbio/proscan/üORFdetectorsüNCBI:üPromoterpredictorsüCSHL:üBDGP:fruitfly.org/seq_tools/promoter.htmlüICG:TATA-BoxpredictorüPolyAsignalpredictorsüCSHL:argon.cshl.org/tabaska/polyadq_form.htmlüSplicesitepredictorsüBDGP:üStart-/stop-codonidentifiersüDNALC:Translator/ORF-FinderüBCM:SearchlauncherOnlineToolsTranscriptionalfactorsbindingsitesmutationanalysisTranscriptionalactivitydetectionusingLuciferasereporterassaysDeterminationofcertaintranscriptionalfactoractivityEMSA:ElectrophoreticMobilityShiftAssayCellnuclearextractorrecombinantproteinEMSA:ElectrophoreticMobilityShiftAssayDNAFootprintassay:invitroandinvivoDNAFootprintassay:invitroandinvivochromatinimmunoprecipitation(ChIP)Post-transcriptionaleventsØmRNAProcessingI:•Capping(加帽)•Polyadenylation(加尾)ØmRNAProcessingII:•Splicing(剪接)ØOtherRNAProcessing:rRNAandtRNARemovalofnucleotidesbybothendonucleasesandexonucleasesØendonucleasestocutatspecificsiteswithinaprecursorRNAØexonucleasestotrimtheendsofaprecursorRNAØThisgeneralprocessisseeninprokaryotesandeukaryotesforalltypesofRNA5’CappingØBeforeRNAchainismorethan20-30ntlongØ5’-endismodifiedbyadditionofa7-met