生物化学讲义Chapter26(complete)RNAMetabolism

Chapter26RNAMetabolism,1.HowisRNAsynthesizedusingDNAtemplates(transcription)?2.HowisnewlysynthesizedprimaryRNAtranscriptsfurtherprocessedtomakefunctionalRNAmolecules?3.HowisRNAandDNAsynthesizedusingRNAastemplate(reversetranscription);4.WhatistheevolutionaryimplicationofthestructuralandfunctionalcomplexityofRNAmolecules?,1.RNAmoleculeshavegreatstructuralandfunctionaldiversity,Withstructurescomparabletoproteinsincomplexityanduniqueness.FunctionasmessengersbetweenDNAandpolypeptides(mRNA),adapters(tRNA)tomatchaspecificaminoacidwithitsspecificgeneticcodecarriedonmRNA,andthestructuralandcatalyticcomponentsoftheprotein-synthesizingribosomes(rRNA).StoresgeneticinformationinRNAviruses.CatalyzestheprocessingofprimaryRNAtranscripts.MighthaveappearedbeforeDNAduringevolution.,2.DNAandRNAsynthesesaresimilarinsomeaspectsbutdifferentinothers,Similarinfundamentalchemicalmechanism:bothareguidedbyatemplate;bothhavethesamepolarityinstrandextension(5to3);bothusetriphosphatenucleotides(dNTPorNTP).Differentaspects:Noprimersareneeded;onlyinvolvesashortsegmentofalargeDNAmolecule;usesonlyoneofthetwocomplementaryDNAstrandsasthetemplatestrand;noproofreading;subjecttogreatvariation(when,whereandhowefficienttostart).,3.ThemultimericRNApolymeraseinE.colihasmultiplefunctions,Theholoenzymeconsistsoffivetypesofsubunits(a2bbs)anditsisusedtosynthesizealltheRNAmoleculesinE.coli.Themultiplefunctionsinclude:searchesforinitiationsitesontheDNAmoleculeandunwindsashortstretchofDNA(initiation);selectsthecorrectNTPandcatalyzestheformationofphosphodiesterbonds(elongation);detectsterminationsignalsforRNAsynthesis(termination).,TheE.coliRNApolymeraseholoenzymeconsistsofsixsubunits:a2bbs.,Possiblecatalyticsubunits,Promoterspecificity,Enzymeassembly,promoterrecognition,activatorbinding,Roleunknown(notneededinvitro),36.5kDa,151,155,11kDa,(32-90kDa),4.RNAsynthesisoccursina“moving”transcriptionbubbleontheDNAtemplate,OnlyashortRNA-DNAhybrid(8bpinbacteria)ispresentthroughthetranscriptionprocess.Ateachmoment,aregionofabout17bpontheE.coliDNAisunwoundinthetranscriptionbubble.TheRNAchainisextendedatarateof50-90nucleotides/secondbytheE.coliRNApolymerase.UnwindingaheadofandrewindingbehindofthetranscriptionbubbleproducespositiveandnegativesupercoilsrespectivelyontheDNA(relievedbytheactionoftopoisomerases).,5.RNApolymeraserecognizesspecificpromotersequencesonDNAtoinitiatetranscription,Promotersequencesarelocatedadjacenttogenes.Promoterscanbeidentifiedusing“protectionassays”(e.g.,footprintingtechniques).Promoters,althoughallbindtothesamepolymerase,havequitevariableDNAsequences(surprisingly),butwithtwoconsensussequencescenteredat10and35positions(thefirstresidueoftheRNAisgiven+1).Promotershavingsequencesmoresimilartotheconsensusaremoreefficient,andviceversa(fromstudiesofmutationsandactivitycomparison).,Thefootprintingtechnique,Thefootprint,-protein,+protein,randomly,Footprinting:PurifiedRNApolymerase(orotherDNAbindingprotein)isfirstmixedwithisolatedandlabeledDNAfragmentthatisbelievedtobindtotheaddedproteinBeforethatDNAiscutwithnonspecificDNase.,IntheabsenceofDNA-bindingprotein,InthepresenceofDNA-bindingprotein,Anactualfootprintingresult(RNApolymerasebindingtoalacpromoter),Thefootprints,AlignmentofdifferentpromotersequencesfromE.coligenes:the10(thePribnowbox)and35consensusregionwererevealed.,SequencesofthecodingDNAstrandisconventionallyshown,AddTTTACCN12TATAATN7A,Presentonlyincertainhighlyexpressedgenes,PromoterofE.coliAddgene,6.ThessubunitsenabletheE.coliRNApolymerasetorecognizespecificpromotersites,TheRNApolymerasewithoutthessubunit(i.e.,thea2bb)isunabletostarttranscriptionatapromoter.ThessubunitdecreasestheaffinityofRNApolymeraseforgeneral(non-promoter)regionsofDNAbyafactorof104.E.colicontainsmultiplesfactorsforrecognizingdifferentpromoters,e.g.,s70forstandardpromoters;s32forheat-shockpromoters;s54fornitrogen-starvationpromoters.Eachtypeofsfactorallowsthecelltocoordinatelyexpressasetofgenes.,StandardHeat-shockNitrogenstarvation,s70forstandardpromoters;s32forheat-shockpromoters;s54fornitrogen-starvationpromoters,E.colicontainsmultiplesfactorsforrecognizingdifferenttypesofpromoters:,7.RNApolymeraseunwindsthetemplateDNAtheninitiateRNAsynthesis,Theenzymeslidestoapromoterregionandformsamoretightlybound“closedcomplex”.Thenthepolymerase-promotercomplexhastobeconvertedtoan“opencomplex”,inwhicha12-15bpcoveringtheregionfromtheAT-rich10siteto+3siteisunwound.Theessentialtransitionfroma“closed”toan“open”complexsetsthestageforRNAsynthesis,afterwhichthecorepolymerasemovesawayfromthepromoter.,random,8.E.coliRNApolymerasestopssynthesizingRNAatspecificterminatorDNAsequences,Twoclassesoftranscriptionterminatorshavebeenidentifiedinbacteria:onedependsonrprotein,theotherisr-independent.Attherindependentterminator,thetranscribedRNAisabletoformastem-loop(palindromicinDNAsequence)structurefollowedbyastretchofUs(oligoAinDNA).Ther-dependentterminatorneedstherprotein,whichhasanATP-dependentRNA-DNAhelicaseactivity,forstoppingRNAsynthesis.,Ther-dependentterminatorDNAexhibitnoobvioussequencesimilarities(probablytheRNApolymerasedetectsnoncontiguousstructuralfeatures?).Ther-dependentterminatorismoreoftenfoundinphages(whereitwasoriginallydiscovered),butrarelyinE.coli.Incontrasttowhatwasoriginallyexpected,theactivesignalsforstoppingRNAsynthesisinbothr-independentandr-dependenttranscriptionterminatorslieinthenewlysynthesizedRNAratherthanintheDNAtemplate.,r-independentterminator:amodel,PalindromeDNAsequences,OligoUs,Stem-loop(hairpin)structure,TranscriptionterminatorofE.coliAddgene,Modelforanr-dependentterminator,9.Transcriptionisahighlyregulatedprocess,Transcriptionisthefirststepinthecomplicatedandenergy-expensivepathwayleadingtoproteinsynthesis,anidealtargetforregulatinggeneexpression.TheRNApolymerasebindstoeachpromoterinverydifferentefficiency.ProteinfactorsbindingtoDNAsequencescloseordistanttothepromoterscanpromote(activator)orrepress(repressor)thesynthesisofcertainRNAmolecules.,10.ThreekindsofRNApolymerases(I,II,andIII)havebeenrevealedformakingRNAsinthenuclearsofeukaryoticcells,Eachisresponsibleforthetranscriptionofacertaingroupsofgenes:rRNA,mRNAortRNAgenes.Theenzymesareoftenidentifiedbyexaminingtheirsensitivitytowardsa-amanitin(fromatoxicmushroom).,EukaryoticRNApolymerases,11.RNApolymeraseII(PolII)bindstopromotersofthousandsofprotein-codinggenes,ManyPolIIpromoterscontainaTATAAAsequence(calledaTATAbox)at-30positionandaninitiatorsequence(Inr)at+1position.Thepreinitiationcomplex(includingPolII)isbelievedtoassembleattheTATAbox,withDNAunwoundattheInrsequence.However,manyPolIIpromoterslackaTATAorInrorbothsequences!,Generalfeaturesofpromotersforprotein-codinggenesinhighereukaryotes,GCbox,CAATbox,12.PolIIishelpedbyanarraysofproteinfactors(calledtranscriptionfactors)toformanactivetranscriptioncomplexatapromoter,FirsttheTATA-bindingprotein(TBP)bindstotheTATAbox,thenTFIIB,TFIIF-PolII,TFIIE,andTFIIHwillbeaddedinorderformingtheclosedcomplexatthepromoter.TFIIHthenactsasahelicasetounwindtheDNAduplexattheInrsite,formingtheopencomplex.AkinaseactivityofTFIIHwillphosphoryatetheC-terminaldomain(CTD)ofPolII,whichwillinitiateRNAsynthesisandreleasetheelongationcomplex.,TFIIEandTFIIHwillbereleasedaftertheelongationcomplexmovesforwardforashortdistance.ElongationfactorswillthenjointheelongationcomplexandwillsuppressthepausingorarrestofthePolII-TFIIFcomplex,greatlyenhancingtheefficiencyofRNAsynthesis.TheterminationoftranscriptionofPolIIhappensbyanunknownmechanism.ThisbasalprocessofinitiatingRNAsynthesisbyPolIIiselaboratelyregulatedbymanycellortissuespecificproteinfactorsthatwillbindstothetranscriptionfactors,mostlyactinapositiveway.,WhenPolIItranscriptionstallsatasiteofDNAlesion,TFIIHwillbindsatthelesionsiteandappearstorecruittheentirenucleotide-excisionrepaircomplex.,TBP,DNA,AproposedmodelforPolII-catalyzedmRNAsynthesis,13.TheactionofRNApolymerasescanbespecificallyinhibited,Three-ring-containing,planarantibioticmoleculeslikeactinomycinDintercalatesbetweentwosuccessiveGCbasepairsinduplexDNA,preventingRNApolymerases(alltypes)tomovealongthetemplate(thustheelongationofRNAsynthesis).Rifampicin(anantibiotic)bindstothebsubunitofbacterialRNApolymerases,preventingtheinitiationofRNAsynthesis.a-amanitinblockseukaryoticmRNAsynthesisbybindingtoRNApolymeraseII.,14.RNAmoleculesareoftenfurtherprocessedafterbeingsynthesizedontheDNAtemplate,TheprimarytranscriptsofeukaryoticmRNAsareoftencappedatthe5end,splicedinthemiddle(intronsremovedandexonslinked),polyadenylatedatthe3end.TheprimarytranscriptsofbothprokaryoticandeukaryotictRNAsarecleavedfrombothends,splicedinsomecases,andmodifiedformanyofthebasesandsugars.,15.AneukaryoticmRNAprecursoracquirea5capshortlyaftertranscriptioninitiates,AGMPcomponent(fromaGTP)isjoinedtothe5endofthemRNAinanovel5,5-triphosphatelinkage.TheguaninebaseisthenmethylatedattheN-7.The2-OHgroupsofthe1stand2ndnucleotidesadjacenttothe7-methylguaninecapmayalsobemethylatedincertainorganisms.ThemethylgroupsaretransferredfromS-adenosylhomocysteine.,A5capisaddedtoeukaryoticmRNAsBeforetranscriptionends,The5capfoundontheeukaryoticmRNAs,16.MosteukaryoticmRNAshaveapoly(A)tailatthe3end,Thetailconsistsof80to250adenylateresidues.ThemRNAprecursorsareextendedbeyondthesitewherepoly(A)tailistobeadded.AnAAUAAAsequencewasfoundtobepresentinallmRNAsandmarks(togetherwithothersignalsatthe3end)thesiteforcleavageandpoly(A)tailaddition(11to30nucleotidesonthe3endoftheAAUAAAsequence).Thespecificendonucleaseandpolyadenylatepolymerase,andotherproteinsprobablyexistasamultiproteincomplextocatalyzethisevent.,Apoly(A)tailisusuallyaddedatthe3endofanmRNAmoleculeviaaprocessingstep.,17.EMstudiesofmRNA-DNAhybridsrevealedthediscontinuityofeukaryoticgenes,EachgenewasfoundtobeacontinuousfragmentofDNAinthebacterialgenome.ButBergetandSharp(1977)observedsingle-strandedDNAloopswhenexaminingadenovirusmRNA-DNAhybridsbyelectronmicroscopy.Suchsingle-strandedDNAloopswaswidelyobservedwhenexaminingsuchRNA-DNAhybrids.IntronsequenceswereproposedtobepresentonthetemplateDNAsequences,whichareremovedduringRNAprocessing,withexonslinkedtogetherprecisely.,Almostallgenesinvertebratescontainintrons(buthistonegenesdoesnot).Manygenesincertainyeastsdonotcontainintrons.Intronsarealsofoundinafewbacterialandarchaebacterialgenes(butfarlesscommonthanineukaryoticcells).,EMstudiesofmRNA-DNAhybridsforthechickenovalbumingene(theR-loopingtechnique),18.Fourclassesofintronshavebeenrevealedhavingdifferentsplicingmechanisms,GroupIintronsarefoundinsomenuclear,mitochondrialandchloroplastgenesencodingrRNAs,mRNAs,andtRNAs.GroupIIintronsareoftenfoundingenesencodingmRNAsinmitochondrialandchloroplastDNAoffungi,algae,andplants.GroupIIIintrons(thelargestgroup)arefoundingenesencodingeukaryoticnuclearmRNAs.GroupIVintronsarefoundingenesencodingthetRNAsinthenucleargenomicDNAofeukaryotes,19.GroupIintronsareself-splicinganduseaguaninenucleosideornucleotideasthecofactor,TheintronpresentintherRNAprecursorofTetrahymenawasfoundtoberemovedbyitselfwithoutusinganyproteins(ThomasCech,1982).Theintronisremovedandthetwoexonspreciselylinkedviatwonucleophilictransesterificationreactions(withtwo3-OHgroupactasthenucleophiles).,GroupIintronsareremovedbyself-splicingviatwonucleophilictransesterificationreactions.,Thepredictedsecondarystructureoftheself-splicingrRNAintronofTetrahymena,Theinternalguidesequence,5splicesite,3splicesite,20.GroupIIintronsalsoundergoself-splicingusingformingalariat-likeintermediate,Butthe2-OHgroupofanadenylateresiduewithininremovingintronplayedtheroleofthe3-OHgroupoftheguaninenucleosideornucleotideingroupIintronself-splicing.,GroupIIintronsareremovedviaself-splicingwithanadenylateresidueoftheremovingintronactsasthenucleophile,forminganlariate-likeIntermediate.,21.TypeIIIintronsarefoundinthenuclearmRNAprimarytranscriptsandhavethelargestnumbers,Thesplicingexon-intronjunctions,determinedbycomparingthesequencesofthegenomicDNAwiththatofthecDNApreparedfromthecorrespondingmRNA,inmRNAprecursorsarespecifiedbysequencesatthetwoendsoftheintrons:beginwithGUandendwithAG.TypeIIIintronsareremovedviaaverysimilarwayasthatoftypeIIintronsexceptbeinghelpedbyseveralhighlyconservedsmallnuclearribonucleoproteins(snRNPs),eachcontainingaclassofU-richsmallnuclearRNAs(snRNAs).,TypeIIIintrons,foundonnuclearmRNAprimarytranscripts,areremovedviathespliceosomes,22.groupIVintronsarefoundintRNAprecursorsandareremovedbyendonucleaseandRNAligase,Thesplicingendonucleasefirstcleavesthephosphodiesterbondsatbothendsoftheintron.ATPisneededfortheRNAligaseactivitytojointhetwoexons.ThejoiningreactionissimilartotheDNAligase-catalyzedreaction.ThemechanismofcleavinggroupIVintronsisdifferentfromthatofgroupI,II,andIIIintrons,allincludingtwotransesterificationreactions.,GroupIVintronsaresplicedviatheactionofspecificendonucleaseandRNAligase.,RNAligase,23.AlternativeproteinsmaybeproducedfromonesinglegeneviadifferentialRNAprocessing,Themultipletranscriptsproducedfromsuchagenemayhavemorethanonesiteforcleavageandpolyadenylation(asforimmunoglobulinheavychains),alternativesplicing(asforthemyosinheavychainsinfruitflies),orboth(asforthecalcitoningeneinrats).Indifferentcellsoratdifferentstagesofdevelopment,thetranscriptmaybeprocesseddifferentlytoproducedifferentgeneproducts(proteins).,MultiplemRNAs(thuspolypeptidechains)canbeproducedviadifferentialRNAprocessing.,24.ThedifferentrRNAmoleculesofbothprokaryotesandeukaryotesaregeneratedfromsinglepre-rRNAs,The16S,23Sand5SrRNAs(togetherwithcertaintRNAs)inbacteriaareallgeneratedfromasingle30Spre-rRNA(about6.5kb,transcribedbyRNApolymeraseI).Therearesevenpre-rRNAgenesintheE.coligenome(eachencodingadifferenttRNA).The18S,28Sand5.8SrRNAsineukaryotesaregeneratedfromasingle40Spre-rRNA(14kb).The5SrRNAineukaryoticcellsisgeneratedseparately(transcribedbyRNApolymeraseIII).,AlltherRNAsarederivedfromasingleprecursorinprokaryoticcells.,The18S,5.8S,and28SrRNAsineukaryoticcellsarederivedfromonepre-rRNAmolecule(theprocessingneedssmallnucleolarRNA-containingproteins).,25.PrimarytRNAtranscriptsundergoaseriesofposttranscriptionalprocessing,Theextrasequencesatthe5and3endsareremovedbyRNasePandRNaseDrespectively.TheRNAinRNasePiscatalytic(Altman,1983)TypeIVintronsareoccasionallypresentinpre-tRNAsineukaryoticcells.TheCCAsequenceisgeneratedatthe3endbytheactionoftRNAnucleotidyltransferase(havingthreeactivesitesforthethreeribonucleotidesadded).SomeofthebasesintRNAmoleculesaremodifiedbymethylation,deamination,reductionandothers.,TheprocessingoftheprimarytRNAtranscriptsincluderemovalofthe5and3ends,additionoftheCCAsequenceatthe3end,modificationofmanybases,andsplicingofintrons(ineukaryoticcells).,SometypicalmodificiedbasesfoundInmaturetRNAmolecules.,26.MoreRNAmolecules(ribozymes)werefoundtobecatalytic,CatalyticRNAmoleculeswerealsofoundinthevirusoidRNA(calledhammerheadribozymes).RNAsinthespliceosomes(theU-richRNAs)andribosomesarealsobelievedtobecatalytic.Aspecific3-Dstructureisrequiredforribozymestobecatalytic.Ribozymesoftenorienttheirsubstratesviabasepairing.,Theexcisedintron(414nucleotides)ofthepre-rRNAofTetrahymenaisfurtherprocessedtoaRNAfragmentof395nucleotidesnamedasL-19IVS;(interveningsequencelacking19nucleotides)Aportionoftheinternalguidesequenceremainsatthe5endofL-19IVSandtheguanosinebindingsiteisstillintact.Dr.CechreasonedthatL-19IVSmightactonexternalsubstrates.L-19IVSisabletocatalyzethelengtheningofsomeoligonucleotides,likea(C)5oligomer,attheexpenseofothers(beingbothanucleaseandpolymerase).,L-19IVSfunctionsasarealcatalystinthetesttube,Incubationtime(minutes),Labeledsubstrate,RNANucleaseactivity,RNApolymeraseactivity,TheM1RNAinribonucleasePiscatalytic,Theintroninthepre-rRNAofTetrahemenaisself-spliced,27.ThecellularmRNAsaredegradedatdifferentrates,ThelevelofaproteininacellisdeterminedtosomeextentbythelevelofitsmRNA,whichdependsonabalanceoftheratesonitssynthesisanddegradation.ThehalfoflivesofdifferentmRNAmoleculesvarygreatly,fromsecondstomanycellgenerations.3hairpinandpoly(A)tailshavebeenshowntoincreasehalvelivesofmRNAs,butmultiple,sometimesoverlappingAUUUAsequenceshavebeenshowntodecreasehalvelives.,The53exoribonucleaseisprobablythemajordegradingenzymeformRNAs.ThepolynucleotidephosphorylasemaybeanotherenzymedegradingmRNAs.PolynucleotidephosphorylasewasusedtosynthesizeRNAforthefirsttimeinthetesttube(SeveroOchoasharedtheNobelPrizewithArthurKornbergin1959forthisdiscovery).(NMP)n+1+Pi(NMP)n+NDPThisenzymewasusedtosynthesizeRNApolymersofdifferentsequencesandfrequenciesofbasesfortheelucidationofthegeneticcodes.,AveragehalflivesofmRNAmoleculesBacteria1.5minutesVertebrates3hours,28.ReversetranscriptasescatalyzetheproductionofDNAfromRNA,Theexistenceofthisenzymeinretroviruses(RNAviruses)waspredictedbyHowardTeminin1962,andprovedbyTeminandDavidBaltimorein1970.Thisenzymecatalyzesthreereactions:RNA-directedDNAsynthesisusingtRNAsasprimers;DegradationoftheRNAtemplate;DNA-directedDNAsynthesis;Theenzymehasno35proofreadingexonucleaseactivity,thusgeneratinghighrateofmutations.ThisenzymeiswidelyusedtosynthesizecomplementaryDNAs(cDNAs)frommRNAs.,ReversetranscriptasescatalyzesthesythesisofDNAfromRNAtemplate.,29.Telomerasecatalyzesthesynthesisoftherepeatingtelomeresequences(TxGy)usinganinternalRNAtemplate,TelomeresconsistofafewtoalargenumberoftandemcopiesofashortoligonucleotidesequencethatarelocatedatthetwoendsofthelinearchromosomalDNAs,havinga3singlestrandextension(ontheTGstrand).Telomeraseactstopreventthechromosomalendsfrombecomingshortenedaftereachreplication(theendpartofthelaggingstrandcannotbeduplicated).,Telomeraseisactuallyareversetranscriptase,butusesashortsegmentofaninternalRNAmolecule(150nucleotides)asthetemplatetoextendtheend.TheCyAxstrand(thelaggingstrand)isbelievedtobesynthesizedbyaDNApolymeraseusingaRNAprimer.Theendsofalinearchromosomeisoftenprotectedbybindingtospecificproteins,formingaTloopstructureinhighereukaryotes,wherethesingle-strandedDNAissequestered.Thelengthofthetelomereseemstobeinverselyrelatedtothelifespanofcellsandindividuals(shortensasoneages).,ProblemposedinthereplicationoflinearDNA:theendofonedaughterstrandwillbeshortenedaftereachroundofreplication.,The“inchworm”(尺蠖）modelfortelomeraseaction,TheTloopobservedatoneen