分子期末復(fù)習(xí)

ChapterTransmissionGenetics傳遞遺傳學(xué)naturalselection自然選擇SurvivaloftheFittest適者生存Allele等位Principleof Principleofindependentassortmentbination遺傳重組geneticma遺傳圖譜geneticlinkage遺傳連鎖one-gene-one-enzyme：eachgeneistranslatedintoanenzymetoperformtaskswithinanorganismPhage噬菌體Transposableelements轉(zhuǎn)座元件doublehelixstructure雙螺旋結(jié)構(gòu)Monoclonalantibody單克隆抗體HGPHumanGenomeProjectTransgenicplants轉(zhuǎn)植物Microarray微陣列ShotgunsequencingcentralDogmaTranscriptionTranslation真核：linear，multipleorigins有omeres（端粒）DeoxyribonucleicAcidDNAPolynucleotides(DNAandRNA)contains:(deoxy)ribose,phosphoricacid;basesBasesincludes:(-Hasdirectionor--BackbonecomprisedofchargedPhosphateresidues-SugarsLinkedbya-phosphodiesterbondLinkthe5‘methylgrouptothe3’OHgroupGenome:theDNAofahaploidsetofchromosomesfromthatDNAdenaturation(melting):thedoubledstrandsof yseparatedeachother. thetemperatureatwhichtheshiftinabsorbanceishalfcompleted.MoreGC,higher uniqueDNAinasample.AhighconcentrationofDNAincubatedforashortertime=alowconcentrationofDNAincubatedforalongertimemitochondria線粒體andchloroplasts(葉綠體)：haveaDNAgenomeorchromosome)，與原核生物相似，endosymbioticorigin（內(nèi)）CvalueSizeofhaploidgenome=C-ChromosomeNucleosome核小體（200bpDNA+Histone八聚體HistoneH1 DNAbetween Filament>30-nmfiberGelelectrophoresisUseconstantcurrent（恒定電流）DNA分子量很大時(shí)線性關(guān)系不再(：accordingThereare3majorstepsinaPCR,whicharerepeatedfor30or40cycles.Thisisdoneonanautomatedcycler,whichcanheatandcoolthetubeswiththereactionmixturei yshorttime.第一步：Denaturationat94C:doublestrandopenstosinglestrandedDNA第二步：Annealingat50-60CprimersbindtotheDNAExtensionat72C:theidealworkingtemperatureforthepolymerase.Thebases(complementarytothetemplate)arecoupledtotheprimeronthe3'sideSequenceinformationoftheGOI（geneofinterest）fordesigningprimersAppropriatetemplate(DNA)Mg2+Buffer(pHandsaltdNTP(materialsformakenewDNA非序列特異性熒光SYBRGreen混合在PCR反應(yīng)液中的熒光探針只有與雙鏈DNA隨著新合成DN段的增加，由于結(jié)合到DNA上的熒光探針增加，被激發(fā)產(chǎn)生的熒光相與目的DNA波長(zhǎng)兩個(gè)不同熒光基團(tuán)，由于彼此距離靠近，在熒光能量轉(zhuǎn)移（FRET）作用下發(fā)生熒隨著PCR反應(yīng)的進(jìn)行，TaqMan探針結(jié)合到目的DNA序列上，并且會(huì)被具有外切酶活性的TaqDNA聚合酶逐個(gè)切除而降解。切下來(lái)的熒光基團(tuán)解除了熒光淬滅的束縛，會(huì)在激發(fā)光下發(fā)出熒光，因此，產(chǎn)生的熒光強(qiáng)度直接反映了所擴(kuò)增靶DNA的總量。比較各樣本之間待檢測(cè)靶DNA多少，顯示的是各樣本之間的相對(duì)含量。利用標(biāo)準(zhǔn)曲線，可以確定樣品中待檢測(cè)靶DNA的絕對(duì)含量。RestrictionEndonucleases限制性?xún)?nèi)切酶切外源DNA（dsDNA）（1）REsthatrecognizespecificnucleotidesequencesandcleavebothstrandsoftheDNAcontainingthosesequences.（2）Recognitionsequencesformanyenzymesarethesameonbothstrands.Suchrecognitionsequencesaresaidtobepalindromic(回文序列）.（3）CommonlyusedREsalwayscleavetheDNAstrandsatafixedpositionrelativetotherecognitionsequence.（4）bluntendsorSticky():(點(diǎn)后不能被限制性酶切（即使只有半條鏈有，會(huì)在后自動(dòng)加上去CH3）DpnI:cutonlymethylatedDNA（進(jìn)化出對(duì)應(yīng)的機(jī)制 Antibioticmarkergene:MCSMultiplecloningsite,多克隆位點(diǎn)allowtheinsertionofforeignDNATypesofvectors.(3)Artificialchromosomes（人工）幾百kbPlasmid：雙鏈環(huán)狀DNA（細(xì)菌中，真核中也有）small，只攜帶幾個(gè)，一個(gè)ori機(jī)制和相同PlasmidDNAreplicationratethatofthehostmultipleplasmids/cellPlasmidDNAreplicationrate=thatofthehostsingleplasmids/cellReplicaplating（平板影印法）（LacZ）acZ肽鏈，通過(guò)互補(bǔ)，能將X-gal變成藍(lán)色。LacZ’中含MCS，外源片段的使LacZ’失活，Λ噬菌體transduceDNAfromonebacterialcelltoanother.Advantagesoverplasmids:（2）攜帶外源(canbeusedformakelreplacingtheintegration-excision(I/E)region（填充片段）withGOI,itcouldforcephagesgettingintolyticcycle(裂解循環(huán))andavoidfromlysogenicstate(溶源循環(huán))，形成大量的噬菌Cosmids(cossite-carryingplasmid(粘粒、柯斯載體)engineeredλPhages,can modateupto50kbinserts.含有λ噬菌體的cos質(zhì)粒的ORI（像質(zhì)粒一樣能400kb，由omeric,centromeric,andreplicationoriginsequencesneededforreplicationinyeastcells.:protectsthelinearDNAfromdegradationbynucleases.CEN:著絲粒著絲點(diǎn)ARS(autonomousreplicatingsequence):自主序列specificDNAsequencesthatallowtheDNAreplicationmachinerytoassembleontheDNAandmoveatthereplicationforks.100to300kbinsertsize(averageof150kb)inE.coliE.Coli的F質(zhì)粒改造而來(lái)EnzymesusedincloningRestrictionenzymeAlkalinephosphatase（堿性磷酸酶）5’磷酸基團(tuán)，防止載體自連DNALigaseOtherDNApolymeraseI:DNAterminitoblunt平endssynthesisofDNAinthe5′->3′direction5′→3′exonucleasefunction.（切除引物，和已存在的DNA）Klenowfragment（5’->3’活性部分的polI）5’突出補(bǔ)平，3’切平EnzymesformakinNAlibraryRNaseH:酶解與DNA配對(duì)的RNA鏈Terminaltransferase（末端轉(zhuǎn)移酶）:addoligo(dC)to softhecDNA3’cDNAannealtocomplementaryoligo(dG)endsofasuitablevectorRT-PCRtocloneasinglecDNA特異性引物：reverseprimeUseareverseprimewithHindIIIsiteatits5-endtostartfirst-strandcDNAPCRreactionusingthesynthesizedcDNAastemplate(forwardprimerhasBamHICutthePCRproductswithHindIIIand ligateitintoaRACE:rapidamplificationofcDNAends,amethodforextendingapartialcDNAtoits5’or3’-end(通過(guò)PCR進(jìn)行cDNA末端快速克隆的技術(shù))TomakethewholelengthofcDNA:5’-RACEand3’-RACEHybridizethe pletecDNAtomRNA,useRTtoextendthecDNAtothe5’-endoftheAddCresidualstothe3’-endoftheextendedcDNA(terminalUseanoligo(dG)primertosynthesizethesecondstrandofPerformPCRusingknown3’sequenceandoligo(dG)asprimersAsimilarproceduretoextendthecDNAinthe3′-directionUsingananchorprimer(錨定引物)(已知序列）Inthatcasethereisnoneedtotailthe3′-endofthecDNAwithterminaltransferasebecausethemRNAalreadycontainspoly(A)andtheanchorsequence;thus,thereverseprimerwouldbetheanchorprimer.（A-T結(jié)合不穩(wěn)定）MethodsofexpressingclonedTomakealargetyofte’s使用cDNA(內(nèi)含子被移除CouldusingantibodytoidentifyclonesfromtheexpressinglibraryExpressionvectors表達(dá)載體PBAD(apromoterfromaraoperon):(TheexpressionproductscontainextraaminoThefusionproteincanbeeasilypurifiedusingaffinitychromatography親和層析法（His）6Ni穿梭載體：在E.coli中，在真核中表達(dá)酵母yeast桿狀 Tiplasmid土壤農(nóng)桿菌（針對(duì)植物）TDNA會(huì)整合到植物上。形成Crowngall(冠狀腫瘤)Left（right）bordergenesforplanttumorigenesisauxinandcytokininbiosynthesis)促進(jìn)細(xì)胞生長(zhǎng)生產(chǎn)瘤andmakingspecialAAfortheirsurvive(opine)冠癭堿Geneofinterestorreportergene(GOI)Multiplecloningsites(MCS)GeneticmarkersforselectionofclonesinplantsOutsideofT-DNA:Geneticmarkersforselectionofclonesinbacteria;等電點(diǎn)梯度（水平）在等電點(diǎn)pH附SDSStep1:themixtureofproteinsiselectrophoresedthroughanarrowtubegelcontainingmoleculescalledampholytes(雙性電解質(zhì))thatsetupapHfromoneendofthetubetotheother.Anegativechargedmoleculewillelectrophoresestowardstheanodeuntilitreachesitsisoelectricpoint(等電點(diǎn)).Step2:Thegelseparatedfromstep1isplacedatthetopofaslabgelforordinarySDS-ProteinswillberesolvedaccordingtotheirPeptidemassNucleicacidhybridization核酸雜交Southernblot:DNA（對(duì)象）Northernblot:RNA（對(duì)象）SouthernGelelectrophoresisofthedigestedDNADNAfragmentsaredenaturedandtransferredintoanitro-cellulose將解鏈的DNA轉(zhuǎn)到硝酸纖(4）nonspecificDNAprotein封閉膜，Usingcertainlabeledprobestohybridizethe(5）Wishouttheextraconsistsofshort,repeatingCsClcentrifugation在CsClVeryshort(2to20-30bp),intandemarraysConcentratednearthecentromeresand literepeatsarethebasisoftheDNAfingerprinting(印跡)DNAtheuseofhighlyvariableregionsofDNAtoidentifyparticularindividuals,useaminisaliteDNAasaprobeMinisaliteDNA(小DNA):highlypolymorphic多態(tài)的.5-100bplong,upto3000repeats.MicrosaliteDNA(微DNA):2-6bpsequencerepeatedto.50-100bplengths,inhumangenome,about30000loci.scatteredthroughoutgenome.看不到分離的峰 liteCutDNAwithrestrictionGelelectrophoresed,denaturedandTheblotwashybridizedwithalabeled liteDNADetectionofthelabeled根據(jù)內(nèi)切酶位點(diǎn)的多態(tài)性,southernDNA分型RFLP（RestrictionFragmentLengthPolymorphism)限制性?xún)?nèi)切酶片段長(zhǎng)度多態(tài)性基于PCRDNA無(wú)需做Southern,只需對(duì)微位點(diǎn)進(jìn)行PCR擴(kuò)NorthernsimilartoaSouthernblot,butitcontainselectrophreticallyseparationRNAsinsteadofDNAsUseDNAasprobeUsetostudytheexpressionofa需要內(nèi)參（loadingInsitu-hybridization原位雜交)tolocatinggeneinHybridizelabeledprobestowholechromosomestolocategenesorotherspecificDNAsequenceFISH:fluorescenceinsitu-hybridization(熒光原位雜交)Digoxigenin(地高辛 labledDNARabbitantiDigoxigeninantibody1抗(InsituRNAhybridization:HybridizelabeledprobestoparticulartissuestoseetheexpressionofaDNAsequencingSangerChain-terminationsequencingUsedideoxynucleotides (雙脫氧核苷酸）toterminateDNAsynthesisyieldingaseriesofDNAfragmentwhosesizescanbemeasuredbyelectrophoresis將目標(biāo)DNA單個(gè)小片段DNAcuttheDNAinquestionwithtwoormorerestrictionenzymesinseparatereactions,measurethesizesoftheresultingfragmentsTodetermininginwhichorientationtheinsertionwasligatedintotheGenefunctionPromoter-reportergenecassettecsobeusedforhighereukaryoticUsingtransformationtechnology,Promoter-reportergenecassettecanbeexpressedintheByassayingtheexpressionofthereportergene,wecanstudywhere,when,andhowte(regulatedbywho?)isexpressed常見(jiàn)報(bào)告GUS(uidA)(β-glucuronidase,葡萄糖甘酶);luc(luciferase,熒光素酶);GFP(Greenfluorescentprotein,綠色熒光蛋白) ysisofGeneExpression，錨定酶識(shí)別4個(gè)堿基。分離3’端酶解片段并將其分為兩份，分別與連接子1和連接子2（均含有IIS類(lèi)酶的識(shí)別位點(diǎn)）結(jié)合，用酶（一種Ⅱ類(lèi)限制酶，它能在距離識(shí)別位點(diǎn)數(shù)DNA雙鏈）BsmF112的樣品混合來(lái)自轉(zhuǎn)錄物3’端一段21bp的序列不同的IIS類(lèi)酶（MmeI）RNA-Seq:arevolutionarytoolfortranscriptomicstativeRT-PCR(定量RT-PCR) chip殘基對(duì)蛋白質(zhì)的結(jié)構(gòu)、催化活性以及結(jié)合配體能力的影響，也可用于改造DNA調(diào)控元件特Site-directedmutagenesisbyoverlapextension延伸誘變Geneknockout敲除技敲除（geneknock-out）又稱(chēng)打靶，通過(guò)外源DNA與DNA之間的同源重組，進(jìn)行精確的定點(diǎn)修飾和改造，具有專(zhuān)一性強(qiáng)、DNA可與目的片段共同穩(wěn)定遺傳敲除是指通過(guò)定位重組系統(tǒng)實(shí)現(xiàn)特定時(shí)間和空間的敲除。Neor新霉素抗性組DNA導(dǎo)入胚胎干細(xì)胞純系中，使外源DNA與胚胎干細(xì)胞組中相應(yīng)部分發(fā)生同源重組，將重組載體中的DNA序列整合到內(nèi)源組中并得以表達(dá)。ClonedDNAcontainingthemousegenetobeknockedoutisinterruptedwithanothergenethatconferstoneomycin（2）AthymidinekinasegeneisplacedoutsidetheMixengineeredmouseDNAwithstemcellssointerruptedgenewillfindwayintonucleusandhomologous binationwilloccurbetweenthealteredgeneandtheresident,intactgene篩選(1）IntroducetheinterruptedgeneintoawholeInjectengineeredcellsintoamouseblastocystImplanttheembryointoasurrogatemotherwhowillgivebirthtochimericmouseTrueheterozygoteresultswhenchimeramateswithablackmousetoproducebrownmice,halfofwhichwillhaveinterruptedgene胚胎干細(xì)胞（ES細(xì)胞）分離和體外培養(yǎng)的成功奠定了哺乳動(dòng)物敲除的技術(shù)基礎(chǔ)。通用轉(zhuǎn)錄因子：所有轉(zhuǎn)錄都需要（如RNA聚合酶 ReportvectorcontainstheDNAelementofinterestfusedtoaminimalpromoterofYeast(GAL4)infrontofareportergene（HIS3營(yíng)養(yǎng)缺陷）ADvectorcontainsthecDNAencodingpotentialtranscriptionfactorsfusedtoaknownYeasttranscriptionfactorGAL4)whichbindstotheminimalpromoterinBaittoinitiatetranscription去掉了BD（與DNA結(jié)合區(qū)域）結(jié)合上蛋白質(zhì)的DNA在凝膠上的遷移率變?。ǎ┩凰貥?biāo)記或生物素標(biāo)記（1抗2抗辣根過(guò)氧化物酶）Chromatinimmunoprecipitation免疫共沉淀蛋白-DNA間invivo體內(nèi)ChIP超聲波打斷200bpInvitroproteininteractionmethodsPAGE膠上分離好的蛋白樣品轉(zhuǎn)移到尼龍膜上（或表達(dá)文庫(kù)菌落印到尼龍膜哪種蛋白能與標(biāo)記了同位素的誘餌蛋白發(fā)生作用，最后顯影。轉(zhuǎn)膜前需要將蛋白復(fù)性。結(jié)合的蛋白后，可以通過(guò)掃描上的熒光點(diǎn)檢測(cè)到穩(wěn)定的相互作用蛋白點(diǎn)。，導(dǎo)相互獨(dú)立的結(jié)構(gòu)域，其中DNA結(jié)合結(jié)構(gòu)域（binding，BD）和轉(zhuǎn)錄激活結(jié)構(gòu)域Invivoprotein-protein AVisual Protein-ProteinInteractionsinplants protein protein proteinA/B noproteinAprotein proteinAproteinDicer（RNAaseIII活性的核酸酶）RNA（30個(gè)核苷酸以上）21～25個(gè)核苷酸的小分子干擾核糖核酸（siRNA，shortinterferingRNA），并有效地定位目標(biāo)mRNA。由siRNA中的反義鏈參與指導(dǎo)合成被稱(chēng)為RNA誘導(dǎo)的沉默復(fù)合體（RISC）的白體，RISCmRNAsiRNA反義鏈互補(bǔ)的區(qū)域，從而實(shí)現(xiàn)干擾靶DNADensitygradientcentrifugationOkazakifragmentOnestrandisreplicatedcontinuouslyinthedirectionofthemovementofthereplicatingfork;theotherisreplicateddiscontinuouslyas1-2kbOkazakifragmentsintheoppositedirection.PrimerDNApolymerasecannotinitiateDNAsynthesiswithouta10-12ntlongRNADNApolymerasesaretemplate-andprimer-dependent(DNA聚合酶依賴(lài)于DNA模板與引物)AllDNApolymerasessynthesizeDNAinthe5to3direction,readingthetemplate3to5’(所有的DNA聚合酶按5‘-3’方向[模板DNA3‘-5’方向]合成DNA)DNApolymerasesrequirethefourdeoxynucleosidetriphosphates(dATP,dGTP,dCTP,andDnaB（separatetwoparentalDNAstrandsatthereplicatingfork.keptthesinglestrandedDNAseperatedandpromotingtheprogressofthereplicationIntroducetemporarysingle-strandbreaks(nicks)calledTypeItopoisomerases切開(kāi)單鏈再連起來(lái)，無(wú)需ATP能解開(kāi)也能加入supercoiling。需ATP（加入negativesupercoiling）SynthesisoftheRNADNA的引物是RNA。引物RNA由RNA引物酶合成。因RNA合成無(wú)需引物，引物RNA50DNAprimosome（引DNApolymeraseI：RemovingofRNAprimerfillthegapthatRNAprimerleaves3‘-5’5‘-3’核酸外切酶活性exonuclease，5‘-3’DNA聚合酶活性DNApolymeraseIIIholoenzyme全酶：TheenzymecarriesouttheelongationofprimerstomakeboththeleadingandlaggingstrandsofDNA3‘-5’外切酶活性DNAtakesplacewhentheOkazakifragmentsarereadytobelinkedtogether. FirsttheRNAprimerisremovedandDNAsynthesizedinitsplace.reversetranscriptase,inthatithasasmallRNAtemplateasapartoftheenzymestructure.addaomererepeattothe3'endCowiththegapsleftbyprimerremoval環(huán)狀的不需要III合成。前導(dǎo)鏈合成以３‘－５’鏈為模板（按５‘－Ａ，再由ＤＮＡ聚合酶III作用合成ＤＮＡ（Okazaki片段直至遇到下Okazaki片段，RNaseH與ＤＮＡ聚合酶II將缺口補(bǔ)齊，再由ＤＮＡ連接酶將兩個(gè)Okazaki片段連接形成大片段ＤＮＡ。WaysofDNABidirectionalreplication(雙向): Tworeplicatingforksmovinginoppositedirectionsawayfromtheorigin.ORI(起始子):auniqueDNAsequenceatwhichDNAreplicationisinitiated.（子）:alltheDNAreplicatedfromoneoriginofreplicationbeginreplicationatseveraloriginpoints.Eachreplicationoriginproceedsbi-directionally,againwithleadingandlaggingstrandsaccordingtothesamerules.Unidirectionalreplication(單向): withonereplicatingforkmovefromtheotherRollingcirclereplication(滾環(huán)):onestrandofadouble-strandedcircularDNAremainsintact&servesasthetemplateforelongationoftheotherstrandatanick.phageswithsingle-strandedcircularDNAgenomes,suchasΦX174GenestructureandtranscriptioninOperon(元：aunitofgeneexpressioninprokarytoesandwhichincludesstructuralgenesandcontrolelements讓簇協(xié)調(diào)一致的工作Polycistronicmessage多順?lè)醋觓nmRNAbearinginformationfrommorethanonePromoter(啟動(dòng)子):ADNAsequencetowhichRNApolymerasebindspriortoinitiationoftranscription-usuallylocatedjustupstreamofthetranscriptionstartsiteofagene.RNA聚合酶-35box:TTGACA,centeredin-35bpUPelementsomepromoterhasanextraelementin40~-60.有一些有，使表達(dá)水平較高Fissites:bacterialenhancers增強(qiáng)子,whichbindstotranscription-activatorproteinFisTranscriptionInitiationsitepurine，A>G。MostlythereareCandTbasesoneithersideofthestartsite:i.e.CGTorCATThemechanismoftranscriptioninbacteriaRNApolymerase：β,β’σ，αRNApolymeraseholoenzyme(全酶）includesβ’,β,σ&Coreenzyme(酶）:withoutσsubunitσ結(jié)合不緊Withoutσfactor,thecoreenzymelostspecificityoftranscriptionInitiationσ2與-10區(qū)結(jié)合，σ4與-35區(qū)結(jié)合ElongationoftranscriptionNeedcoreenzymeDNAunwinding:βinPhosphodiesterbondformationTerminationoftranscriptionΡ(rho)independentAninvertedrepeatthatallowahairpintoform oftheAstringofTsinthenon-templatestrandthatresultsinastringofweakrU-dAbasepairsholdingthetranscriptiontothetemplatestrand.ρ-dependent依賴(lài)于ρ蛋白的terminatorU，所以需要ρ蛋白幫助，終止RNA合成。Rhodepressedtheelongationrate,butnotρreducesthesizeoftheRNAρReleasesTranscriptsfromtheDNARhohasjoinedtheelongationcomplexbybindingdirectlytoRNAThetranscripthaslengthenedandhasboundtorhoviaarholoadingsite,forminganRNAloop.RNA穿過(guò)Rho中間的洞Thepolymerasehaspausedataterminator.Bycontinuouslyfeedingthetranscriptthroughitself,rhohastightenedtheRNAloopandirreversiblytrappedtheelongationcomplex.RhohasalsobeguntodissociatetheRNA-DNAhybrid,whichwillleadtotranscriptrelease.RegulationoftranscriptioninOperator(區(qū)):ADNAelementfoundinprokaryotesthatbindstightlytoaspecificrepressorandtherebyregulatestheexpressionofadjoininggenes嚴(yán)格的調(diào)控：itwillproducespecificproteinsonlywhentheyarerequiredLacoperon:utilization利用ofthesugarlactoseAraoperon:utilizationofthesugarTrpoperon:synthesisoftheaminoacidThreestructureβ-galactosidase(lacZ):breaklactoseintogalactoseandglucoseGalactosidepermease(lacY):totransportlactoseintocellsLacI:GeneencodingtheLacrepressor(LacR)P:Lacpromoter(bindingsiteforRNApol)O:Lacoperator(bindingsiteforallolactose別乳糖結(jié)合到repressor)CAP結(jié)合cAMPmoreglucose,lessTheLacoperonisactivatedonlywhenglucoseconcentrationislow.selectioninfavorofglucosemetabolism: w/iglucose,lacoperonisoff.LacstructuregenesonlyexpressedNoGlucoseThearaAraCAraCproteinasrepressor,itbindstotheoperatorsintwositesandloopthe210bpRepressionrequiresthebindingoftrpandaporepressor(輔阻遏蛋白)Aporepressorisinactive,onlywhenitbindstotryptophan,a arepressor.Lowtrp,norepressionHightrp,repression2、衰減作用Attenuator:aregionofDNAupstreamofoneormorestructuregenes,whereprematuretranscriptionterminationcanoccurArabinoseactsasPolII：hnRNA（異質(zhì)核RNAmRNA前體）snRNApolIII：ConservedsequencesFoundinnearlyallpromoters（保守序列）Non-conservedsequences:與調(diào)控相關(guān)Corepromoter:TFIIBrecognitionelementTATAUpstreampromoterelement(UPE):GC catboxConserved TATAGCSomeclassIIpromotersrequiretheTATAboxforfunction,butothersneeditonlytopositionthetranscriptionstartsite.UpstreamElementsCATbox:GCbox:ahexamerhavingthesequenceofGGGCGGononestrand,whichoccursinanumberofmammalianstructuralgenepromoter.ItisthebindingsiteforthetranscriptionfactorSp1Itisorientationindependent,butnotpositionAtleastoneoftheseelementsismissinginmostPromotersforhighlyexpressedspecializedgenestendtohaveTATAboxes,promotersforhousekeegenestendtolackthem.Somepromoters(TdT)haveonlyaninitiator,possiblywithaClassIpromoterfromrRNAUCE-107--156,core:-45-20spaceOnecommonfeature:AT-richinitiator(rINR)whichsurroundsthetranscriptionstartsiteTwoessentialelements:coreregion:-45-acoreelementsurroundingthetranscriptionstartsite,andanupstreampromoterelement(UPE)about100bpfartherupstream.Thespacingbetweenthetwoelementsisimportant.ClassIII(DNA序列，與所調(diào)控位于同一上Enhancersactthroughproteinsthatbindtothem.Theseproteinsnamedas:transcriptionfactors,enhancer-bindingprotein,oractivators.TBP=TATAbindingprotein;TAF:TBP-associatedprotein;CGeneralTranscriptionfactors通用轉(zhuǎn)錄因子CPreinitiationcomplex:ThecombinationofRNApolymeraseandgeneraltranscriptionfactorsassembledatpromoterjustbeforetranscriptionbeginsTFIIBbindsnext,protein-DNATFIIFhelpsRNApolymerasebindtoRemainingfactorsbinds:TFIIE&TFIITFIIDismostTFIIFisessentialforthebindingofRNATFIID：TATA-box-bindingprotein(TBP)and8-10TBP-associatedfactors(TAFs）TFIID結(jié)合在minorgroove TBPbendsitby~80°TATA缺少則有Inr+DPE或GCbox，TBPClassIfactorsTwoTFs:core-bindingfactor:SL1(human)orTIF-IB(transcriptioninitiationfactor)Upstream-bindingfactor(UBF)orUpstream-activatingfactor(UAF)ClassIIIfactors(TFIIIA,B,TFIIIAisrequiredforthesynthesisof5sRNA,butnottRNATFIIIBandCarerequiredbyboth5srRNAgeneandtRNAgeneTranscriptionalactivators(SpecificTranscriptionalFactors)Withthehelpoftranscriptionalactivators,theexpressionofactivegenesRecognizesequencesinDNA識(shí)別DNA上特定序ControlgeneexpressioninfollowingExpressedinaspecifictissuespatialExpressedinatspecifictimetemporalRequiremodification(e.g.DNA-binding:wellcharacterizedstructuralZincbZip(leucineAcidicGlutamine Proline DNA-binding:Zincfinger(鋅指)majortwohistidinesandtwocysteinesbindingtothezincion Othermononuclearzincfingermotifscanhavethreeorfourcysteines.DNA-binding:hommain(同源結(jié)構(gòu)域)：majorgroove (螺旋-轉(zhuǎn)角-螺旋)CAP mainscanoperateintandemswithsimilarordifferentDNAbindingsbZIP(leucinezipper，亮氨酸拉鏈)leucineappearseverysevenaminoacidsinaregionoftheproteinbHLH(helix-loop-helix)TheGAL4 GAL4responsiblegenescontainsaGAL4site(enhancer,upstreamactivatingsequences,GAL4bindstoaUASGsasadimerbinuclearzincfingerbindingsixCys/HisresiduesandtwozincAlmostallcontactstoDNAinZnclusterregionsaremadebyproteinmainchainatomsLinkerregiondeterminesthespecificityofZn2C6containproteinsChromatinremodellingfactors染色重塑因子SomesubunitsinteractwithRNAPolII,others-withactivatorshistoneacetylaseactivity需要有SWI5proteinbindstotheenhancersequenceandrecruitschromatinremodellingSWI/SNFdecondenseschromatinandexposeshistonetails.HistoneacetylasesgetrecruitedbyHistonetailsgetacetylatedbySBFactivatorgetsboundtopromoterproximal的Mediatorbindsto andformthepreinitiationEpigeneticcontrolmechanismsMethylationofDNAandhistonesDNAmethylationatCpGMethylationofCpGislandscanblocktranscriptionbytwodistinctDirectblocking TFIIDRecruitmentofhistoneDNAmethylationpatterncanbeinheritedtodaughtercellsHistonemodificationpatternisalsoinheritedtodaughtercells(,bybindingtoligands(nuclearAdditionorremovalofoneorseveralphosphategroupsp53tumorsupressor磷酸化激活Ubiquitinationofsomeactivatorscanhaveanactivatingeffect,butpolyubiquitinationmarksthesesameproteinsfordestruction.Sumoylationofdditionofoneormorecopiesof the101-aapolypeptideSUMOtolysineresiduesstable,butunabletoreachtheirgenes.AcetylationofTFsnuclearreceptorstwoidenticalsubunitsandbindtoinvertedDNAhomodimericnuclearreceptorsIntheabsenceofhormone,nuclearreceptorislocatedincytoplasmUponbindingtohormone,thenuclearreceptorgetstransportedtonucleus,whereitbindstotheresponseelementIntheabsenceofhormone,hnrbindstoDNAresponseelementandrecruitshistonedeacetylases.Transcriptionisblocked.Whenhormonediffusesintothenucleusandbindstohnr,histonedeacetylasegetsreleasedandhistoneacetylasebindsinstead.Transcriptionisactivated.MethodsfordetectingchangesintranscriptionlevelNorthernblotRealtimetativeRT-PCRInsituhybridization(ISH,MethodsfordetectingDNAproteininteractionChromatinimmunoprecipitation(ChIP)Gelshiftassay(Electrophoreticmobilityshiftassay,EMSA)DNaseIfootprintingMosthighereukaryoticgenes,codingformRNAandtRNA,andafewcodingfor