基因表达调控

基因表达调控第1页/共26页知识点1真核细胞基因表达的调控主要发生在四个相对独立的水平上[名词]基因表达调控：细胞使遗传信息在正确时间正确位置进行正确表达或关闭以行使特定功能的调节机制。真核细胞基因表达调控发生在4个彼此相对独立的水平上：1.转录水平：决定某个基因不否会被转录，什么时候转录，转录的频率。2.加式水平：决定初始的RNA转录产物如何剪接和加工为成熟的mRNA。3.翻译水平：决定某种mRNA是否会真正得到翻译，如果能得到翻译，决定翻译的频率和时间。4.翻译后水平：在蛋白被翻译后，选择性激活蛋白或使蛋白失活。第2页/共26页知识点2基因表达在转录水平上调控通用转录因子与结合RNA聚合酶的核心启动子位点结合，起始基础水平的转录。特异转录因子分为激活基因转录的激活型转录因子和抑制转录的抑制型转录因子，与基因的各种调控位点结合，促进或抑制基因转录。激素通过激活或抑制某些特异转录因子（激素受体）而影响基因转录。转录因子通常通过调节组蛋白核心结构而改变核小体和染色质紧密程度，影响通用转录因子和RNA聚合酶对启动子的结合来调节基因表达。激活型转录因子通常有利于导致染色质或组蛋白结构松散的蛋白质发挥作用，如组蛋白乙酰化酶；抑制型转录因子通过会加强促进染色质结构紧密的蛋白质作用，如组蛋白脱乙酰酶。第3页/共26页第4页/共26页Eukaryotictranscriptionofprotein-encodinggenesisahighlyregulatedprocessinwhichnumeroustranscriptionactivatorsrecognizespecificDNAelements,proximalanddistanttopromoters,anddirectassemblyofgeneraltranscriptionfactors,co-activatorcomplexesandRNApolymeraseIIonpromoters(thepre-initiationcomplex,atleast50polypeptides).Tounderstandthemolecularmechanismofactivator-dependenttranscriptioninitiation,oneneedstounderstandthepathwayofassemblyofthepre-initiationcomplex,thestructuralorganizationofthepre-initiationcomplex,andhowtheassemblyandstructureofthepre-initiationcomplexareaffectedbybindingofactivatorstocognateDNAelements–averychallengingtaskfortraditional,ensemble,invitro,methods第5页/共26页Modelofhormone-dependentgeneactivationbytheglucocorticoidreceptor(GR)Intheabsenceofhormone,GRisboundinacomplexwithHsp90inthecytoplasmviaitsligand-bindingdomain(lightpurple).Whenhormoneispresent,itdiffusesthroughtheplasmamembraneandbindstotheGRligand-bindingdomain,causingaconformationalchangeintheligand-bindingdomainthatreleasesthereceptorfromHsp90.ThereceptorwithboundligandisthentranslocatedintothenucleuswhereitsDNA-bindingdomain(orange)bindstoresponseelements,allowingtheactivationdomain(green)tostimulatetranscriptionoftargetgenes.ThenuclearreceptorsuperfamilyAllnuclearhormonereceptorsbindtoDNAaseitherhomodimersorheterodimers,butforsimplicityweshowthemasmonomershere.(A)Thereceptorsallhavearelatedstructure.TheshortDNA-bindingdomainineachreceptorisshowningreen.(B)Areceptorproteininitsinactivestateisboundtoinhibitoryproteins.Domain-swapexperimentssuggestthatmanyoftheligand-binding,transcription-activating,andDNA-bindingdomainsinthesereceptorscanfunctionasinterchangeablemodules.(C)Thebindingofligandtothereceptorcausestheligand-bindingdomainofthereceptortoclampshutaroundtheligand,theinhibitoryproteinstodissociate,andcoactivatorproteinstobindtothereceptor'stranscription-activatingdomain,therebyincreasinggenetranscription.(D)Thethree-dimensionalstructureofaligand-bindingdomainwith(right)andwithout(left)ligandbound.Notethattheblueαhelixactsasalidthatsnapsshutwhentheligand(showninred)binds,trappingtheligandinplace.第6页/共26页第7页/共26页第8页/共26页知识点3加工水平的基因表达调控[名词]mRNA剪接：由于真核细胞基因为不连续基因，包含编码的外显子和不编码的内含子，转录时都被转录，之后将不编码的内含子和不需要的外显子切除，形成成熟的mRNA，这个过程称为mRNA剪接。剪接有2种基因方式：1.组成型剪接，2.选择性剪接。[名词]组成型剪接：将前体mRNA中的内含子剪除，规范地将外显子拼接成成熟mRNA。一个基因通过组成型剪接只产生一种成熟mRNA。[名词]选择性剪接：不仅将内含子剪除，还将外显子选择性剪除，从外显子按照不同的方式拼接在一起。一个基因通常可通过选择性剪接产生多个成熟mRNA，翻译产物氨基酸序列有差别，差别程度有大有小。剪接增强子位于保留与否受到调节的外显子内，当它与调节蛋白结合，这个外显子保留；当它不与调节蛋白结合，则被剪除。第9页/共26页Typesofalternativesplicing.

Inthesegraphics,exonsarerepresentedbyboxesandintronsbylines.Exonregionsincludedinthemessagesbyalternativesplicingarecoloredwhileconstitutiveexonsareshowningray.PromotersareindicatedwitharrowsandpolyadenylationsiteswithAAAAFigure2.Locationsofregionsonthepre-mRNAthatcanaffectalternativesplicing.

Somecombinationoftheseregulatoryregionscanusuallybefound.Weakerconsensussplicesitessurroundingthealternativeexon,exonicregulatoryregionsandintronicregulatoryregionsareindicated.Fromresearchoverthepast20years,somegeneralthemeshaveemergedforalternativesplicingregulation,althoughtheexactmechanismsstillneedtobedetermined.Alternativelysplicedexonsoftenhaveweakconsensussequencesatthe5'and3'endsoftheintrons,suggestingthatadditionalsignalsarerequiredforrecognitionoftheexonbythesplicingmachinery.-actingpre-mRNAsequencesresponsibleforregulationofsplicinghavebeenidentifiedformanygenes.Theseregionsarefoundinexonsorinintronsandcanbeenhancersorsilencersofsplicesiteusage(Figure2).Thesesequencemotifsserveasbindingsitesforproteinfactorsthatcanenhanceorinhibittheabilityofthespliceosometorecognizetheexons.Theexonicelementsnotonlyencodeaminoacidsbutalsoregulatetheirownabilitytobesplicedintothematuremessage.-actingsplicingfactorsthatinteractwithsplicingregulatoryelementsinexonshavebeenidentified.SubsetsoftheSRproteinsbindwithregulatorysequencesimportantforsplicingcontrolHeterogeneousnuclearribonucleoprotein(hnRNP)A/Bfamilymemberscanbindtohigh-affinitysequencesinexonsandinhibitsplicingthroughblockingSRproteinsfrombindingtotheexons

第10页/共26页Alpha原肌球蛋白基因转录产物在不同的细胞中有不同的剪接形式在横纹肌细胞在平滑肌细胞在成纤维细胞在纤维细胞在肝细胞在脑细胞原肌球蛋白初始MRA选择性拼接产生不同的成熟mRNA第11页/共26页Dscamisamemberoftheimmunoglobulinsuperfamilyandcontains10Ig-domains,6fibronectintypeIII(FNIII)domains,asingletransmembranedomain,andanovel374AAcontainingcytoplasmicdomain.TheDscammRNAcomprises24exons.Tandemarraysofalternativeexons4,6,9and17allowthegenerationofvariableIgdomainsequencesforIg2,3&7andtwodifferenttransmembranedomains.Mutuallyexclusivesplicingoccursforexons4,6,9,and17.GenomicandcDNAanalysisrevealedthepotentialofgeneratingsome38,000isoformsofDscam.Variableexonsareshownincolor.GraylinesingenomicDNAandboxesinmRNArepresentconstantexons.DietmarSchmucker,Ph.D.AssociateProfessorofNeurobiology

HarvardMedicalSchool

DepartmentofCancerBiology

Dana-FarberCancerInstituteMylaboratoryusesthemodelorganismDrosophila(fruitfly)tostudymolecularmechanismsthatcontrolthedevelopmentofneuronalconnectivity.Anyassemblyofneuronalcircuits,simpleorcomplex,iscontrolledbyaseriesofspecificmolecularsignalingsystemsthatinstructthedirectionalgrowthofneuronalprocesses(axonsanddendrites).Wecombinegenetic,biochemicalandcellbiologicalapproachestostudyhowneuronalsurfacereceptorscontrolthesesignalingsystemsduringaxonanddendriteguidance,branchingandsynaptictargetselection.Wefocusourdevelopmentalandfunctionalstudiesprimarilyontheanalysisofsensoryneuronsofthevisualandsomato-sensorysystemofflies.第12页/共26页RNA的可变剪接有时会产生功能完全没有联系的蛋白质。甲状腺中经选择性剪接，翻译产物是降钙素calcitonin下丘脑中，经选择性剪接，产物是降钙素基因相关多肽GGRP，与血压的调节和痛觉有关选择性拼接产生不同的成熟mRNA第13页/共26页知识点4翻译水平调控mRNA与多种蛋白质作用，决定是否被翻译和翻译的速度、翻译的时间。1.如果mRNA与抑制蛋白结合，就会失去活性而不被翻译。如未受精卵细胞中的mRNA与抑制性蛋白结合形成“隐蔽”mRNA，或者通过microRNA和siRNA途径降解mRNA，使得mRNA得不到翻译。2.非特异性翻译速度调节机制：影响所有mRNA的翻译，如翻译起始因子eIF2磷酸化，降低起始翻译的活性，从而减慢蛋白质合成速率。3.特异性翻译速率调节机制：只改变特定mRNA的翻译速率，如编码铁蛋白mRAN的翻译速率的调控。4.翻译时间的调控：通过调节mRNA的稳定性而调节翻译的时间，如转铁蛋白受体mRNA半寿期的调节。5.翻译的空间调控：有些mRNA在3’端非翻译区有细胞质定位信息，通过微管和微丝运输并定位在细胞特定区域。第14页/共26页ModelofCPEB-dependentmaskingandactivationofmRNAtranslationinoocytes.Typically,maternalmRNAsinitiallycarryashortpoly(A)tailandtheirCPEmotifisboundbyCPEB.CPEBformsacomplexwithmaskin,whichinturninteractswitheIF4E.Thisconfigurationisthoughttorepresentatranslationallyinactiveor“masked”stateofthemRNA.ThesignalforoocytematurationleadstoaphosphorylationofCPEBandastimulationofbindingbetweenCPEBandCPSF.ThroughfurtherassociationofCPSFwithpoly(A)-polymerase,thisleadstoelongationofthepoly(A)tail(indicatedbythelinewithstar).ThebindingbetweenmaskinandeIF4Eisreduced,possiblyasaconsequenceofpolyadenylation.ThisclearsthewayforefficientrecruitmentofeIF4GthroughbindingtoeIF4EandPABPandactivationoftranslation(indicatedbyafurtherlinewithstar).第15页/共26页ByRNAinterference,shortRNA'scanleadtosilencingtheexpressionofgenesthatcontaincomplementarysequencesintheirmRNA.

Acompexofdouble-strandedRNAiscleavedintoshortfragmentsof21-22basepairsinlengthbytheribonucleaseDicer.

ThefragmentsaresiRNA's(shortinterferingRNA's).

ThesiRNA'sbindtotheRISC(RNA-inducedsilencingcomplex).

OneofthestrandsofsiRNAisdegraded.

Theremainingsingle-strandedsiRNA,complexedwiththeRISCcanthenbindtocomplementarymRNA.

Ifaperfectornearperfectmatch,themRNAiscleaved.

Inadditon,theRISC-siRNAcomplexcanenterthenucleus,bindsthegenomicsequenceandinitiatesaDNAmethylationbasedchromatincondensationinactivationofthegene.第16页/共26页Inarelatedmechanism,microRNAs(miRNAs)aregeneproducts(mRNAs)thatare21-22nucleotidesinlength.

TheprimarymiRNAsaretranscribed,formhair-pinstructuresandarecleavedbyDroshatomakeprecursormicroRNAs(roughly70nucleotidesinlength).

Thepre-miRNAsareexportedtothecytoplamwheretheyarecleavedbydicerintothe21-22nucleotidematuremicroRNA's.

ThemiRNA'sformribonucleoproteincomplexeswithmRNA's.

Ifthematchisexact,themRNAisdestroyed,similartosiRNAmechanisms.

Ifthematchisless-than-exact,thenbinding(usuallyofseveralmiRNA's)inhibittranslation.

GenesformiRNA'sseemtomakeup0.5-1.0%ofthetotalnumberofgenesinmulticellularorganisms.

i.e.200-250miRNAgenesinhumans.第17页/共26页ThetranslationinitiationfactoreIF2isaheterotrimericcomplexthatisresponsibleforbindingtheinitiatormethionyltRNA(Met-tRNAiMet)tothesmallribosomalsubunit.ThegsubunitofeIF2containsaclassicGTP-bindingdomain,andGTP-bindingisessentialforbindingMet-tRNAiMettoformtheternarycomplexofeIF2,GTPandMet-tRNAiMet.DuringthecourseoftranslationinitiationtheGTPboundbyeIF2ishydrolyzedtoGDPandeIF2isreleasedfromtheribosomeinabinarycomplexwithGDP.AseIF2hasamuchhigheraffinityforbindingGDPthanGTP,aguanine-nucleotideexchangefactor(GEF)termedeIF2BisrequiredtorecycleeIF2•GDPtoeIF2•GTP.

第18页/共26页Inresponsetoenvironmentalstresses,afamilyofproteinkinasesphosphorylateeIF2(eukaryoticinitiationfactor2)toalleviatecellularinjuryoralternativelyinduceapoptosis.PhosphorylationofeIF2reducesglobaltranslation,allowingcellstoconserveresourcesandtoinitiateareconfigurationofgeneexpressiontoeffectivelymanagestressconditions.Accompanyingthisgeneralproteinsynthesiscontrol,eIF2phosphorylationinducestranslationofspecificmRNAs,suchasthatencodingthebZIP(basicleucinezipper)transcriptionalregulatorATF4(activatingtranscriptionfactor4).ATF4alsoenhancestheexpressionofadditionaltranscriptionfactors,ATF3andCHOP(CCAAT/enhancer-bindingproteinhomologousprotein)/GADD153(growtharrestandDNA-damage-inducibleprotein),thatassistintheregulationofgenesinvolvedinmetabolism,theredoxstatusofthecellsandapoptosis.ReducedtranslationbyeIF2phosphorylationcanalsoleadtoactivationofstress-relatedtranscriptionfactors,suchasNF-kB(nuclearfactorkB),byloweringthesteady-statelevelsofshort-livedregulatoryproteinssuchasIkB(inhibitorofNF-kB).WhilemanyofthegenesinducedbyeIF2phosphorylationaresharedbetweendifferentenvironmentalstresses,eIF2kinasesfunctioninconjunctionwithotherstress-responsepathways,suchasthoseregulatedbymitogen-activatedproteinkinases,toelicitgeneexpressionprogrammesthataretailoredforthespecificstresscondition.LossofeIF2kinasepathwayscanhaveimportanthealthconsequences.MicedevoidoftheeIF2kinaseGCN2[generalcontrolnon-derepressible-2orEIF2AK4(eIF2akinase4)]showsensitivitytonutritionaldeficienciesandaberranteatingbehaviours,anddeletionofPEK[pancreaticeIF2akinaseorPERK(RNA-dependentproteinkinase-likeendoplasmicreticulumkinase)orEIF2AK3]leadstoneonatalinsulin-dependentdiabetes,epiphysealdysplasiaandhepaticandrenalcomplications.第19页/共26页TheintracellularlocalisationofmRNAsisageneralmechanismtotargetproteinstotheregionsofacellwheretheyarerequired,andplaysanimportantroleinthepolarisationofmanycelltypes.AstrikingexampleofthisphenomenonisprovidedbytheDrosophilaoocyte,wherethelocalisationofbicoid,oskar,andgurkenmRNAstothreedistinctpositionswithinthecelldeterminesthepolarityoftheanterior-posterioranddorsal-ventralaxesoftheembryo.UsingthepowerfulgeneticsofDrosophila,weareusingacombinationofmolecular,cell-biologicalandgenetictechniquestoinvestigatethemechanismofmRNAlocalisation.Inaddition,wearestudyinghowthetwoaxesoftheoocytebecomepolarisedtodefinethedestinationofthesetranscripts,inordertounderstandtheoriginofpolarityinDrosophiladevelopmentThedrosophilaisatypeoffruitfly,awell-establishedgeneticmodel.第20页/共26页Translationofferritinisactivatedinthepresenceofiron.

TranslationisinhibitedbybindingoftheIRE-bindingproteintothehairpinstructureofanironresponseelement(IRE)inthe5primeuntranslatedleadersequenceofferritinmRNA.

WhenironbindstoIRE-bindingprotein,itcontortsintoaconformationthatdoesnotrecognizetheIRE.

Whenironisavailable,ribosomescanassembleonthemRNAandproceedtotranslateferritin.

Thehairpindoesnotinterferewiththeribosomeactivities第21页/共26页DegradationofthetransferrinreceptormRNA(requiredforironuptake)isalsoregulatedbytheallostericIRE-bindingprotein.

TransferrinreceptormRNAhasanIREinits3primeuntranslatedregion.

Whenintracellular[iron]islow,theIRE-bindingproteinremainsboundtotheIRE