生物化学讲义Chapter 26 (complete) RNA Me

Chapter26RNAMetabolism1.HowisRNAsynthesizedusingDNAtemplates (transcription)?2.HowisnewlysynthesizedprimaryRNA transcriptsfurtherprocessedtomake functionalRNAmolecules?3.HowisRNAandDNAsynthesizedusingRNA astemplate(reversetranscription);4.Whatistheevolutionaryimplicationofthe structuralandfunctionalcomplexityofRNA molecules?1.RNAmoleculeshavegreatstructuralandfunctionaldiversityWithstructurescomparabletoproteinsincomplexityanduniqueness.FunctionasmessengersbetweenDNAandpolypeptides(mRNA),adapters(tRNA)tomatchaspecificaminoacidwithitsspecificgeneticcodecarriedonmRNA,andthestructuralandcatalyticcomponentsoftheprotein-synthesizingribosomes(rRNA).StoresgeneticinformationinRNAviruses.CatalyzestheprocessingofprimaryRNAtranscripts.MighthaveappearedbeforeDNAduringevolution.2.DNAandRNAsynthesesaresimilarinsomeaspectsbutdifferentinothersSimilarinfundamentalchemicalmechanism:bothareguidedbyatemplate;bothhavethesamepolarityinstrandextension(5`to3`);bothusetriphosphatenucleotides(dNTPorNTP).Differentaspects:Noprimersareneeded;onlyinvolvesashortsegmentofalargeDNAmolecule;usesonlyoneofthetwocomplementaryDNAstrandsasthetemplatestrand;noproofreading;subjecttogreatvariation(when,whereandhowefficienttostart).3.ThemultimericRNApolymeraseinE.colihasmultiplefunctionsTheholoenzymeconsistsoffivetypesofsubunits(a2bb’

s)anditsisusedtosynthesizealltheRNAmoleculesinE.coli.Themultiplefunctionsinclude:searchesforinitiationsitesontheDNAmoleculeandunwindsashortstretchofDNA(initiation);selectsthecorrectNTPandcatalyzestheformationofphosphodiesterbonds(elongation);detectsterminationsignalsforRNAsynthesis(termination).TheE.coliRNApolymeraseholoenzymeconsistsofsixsubunits:a2bb’

s.PossiblecatalyticsubunitsPromoterspecificityEnzymeassembly,promoterrecognition,activatorbindingRoleunknown(notneededinvitro)36.5kDa15115511kDa(32-90kDa)4.RNAsynthesisoccursina“moving”transcriptionbubbleontheDNAtemplateOnlyashortRNA-DNAhybrid(~8bpinbacteria)ispresentthroughthetranscriptionprocess.Ateachmoment,aregionofabout17bpontheE.coliDNAisunwoundinthetranscriptionbubble.TheRNAchainisextendedatarateof50-90nucleotides/secondbytheE.coli

RNApolymerase.UnwindingaheadofandrewindingbehindofthetranscriptionbubbleproducespositiveandnegativesupercoilsrespectivelyontheDNA(relievedbytheactionoftopoisomerases).5.RNApolymeraserecognizesspecificpromotersequencesonDNAtoinitiatetranscriptionPromotersequencesarelocatedadjacenttogenes.Promoterscanbeidentifiedusing“protectionassays”(e.g.,footprintingtechniques).Promoters,althoughallbindtothesamepolymerase,havequitevariableDNAsequences(surprisingly),butwithtwoconsensussequencescenteredat

–10and–35positions(thefirstresidueoftheRNAisgiven+1).Promotershavingsequencesmoresimilartotheconsensusaremoreefficient,andviceversa(fromstudiesofmutationsandactivitycomparison).ThefootprintingtechniqueThefootprint-protein+proteinrandomlyFootprinting:PurifiedRNApolymerase(orotherDNAbindingprotein)isfirstmixedwithisolatedandlabeledDNAfragmentthatisbelievedtobindtotheaddedproteinBeforethatDNAiscutwithnonspecificDNase.IntheabsenceofDNA-bindingproteinInthepresenceofDNA-bindingproteinAnactualfootprintingresult(RNApolymerasebindingtoalac

promoter)ThefootprintsAlignmentofdifferentpromotersequencesfromE.coli

genes:the–10(thePribnowbox)and–35consensusregionwererevealed.SequencesofthecodingDNAstrandisconventionallyshownAddTTTACCN12

TATAATN7A

PresentonlyincertainhighlyexpressedgenesPromoterofE.coli

Addgene6.ThessubunitsenabletheE.coliRNApolymerasetorecognizespecificpromotersitesTheRNApolymerasewithoutthessubunit(i.e.,thea2bb’

)isunabletostarttranscriptionatapromoter.ThessubunitdecreasestheaffinityofRNApolymeraseforgeneral(non-promoter)regionsofDNAbyafactorof104.E.coli

containsmultiplesfactorsforrecognizingdifferentpromoters,e.g.,s70forstandardpromoters;s32forheat-shockpromoters;s54

fornitrogen-starvationpromoters.Eachtypeofsfactorallowsthecelltocoordinatelyexpressasetofgenes.StandardHeat-shockNitrogenstarvations70forstandardpromoters;

s32forheat-shockpromoters;

s54fornitrogen-starvationpromotersE.colicontainsmultiplesfactorsforrecognizingdifferenttypesofpromoters:7.RNApolymeraseunwindsthetemplateDNAtheninitiateRNAsynthesisTheenzymeslidestoapromoterregionandformsamoretightlybound“closedcomplex”.Thenthepolymerase-promotercomplexhastobeconvertedtoan“opencomplex”,inwhicha12-15bpcoveringtheregionfromtheAT-rich–10siteto+3siteisunwound.Theessentialtransitionfroma“closed”toan“open”complexsetsthestageforRNAsynthesis,afterwhichthecorepolymerasemovesawayfromthepromoter.random8.

E.coliRNApolymerasestopssynthesizingRNAatspecificterminatorDNAsequencesTwoclassesoftranscriptionterminatorshavebeenidentifiedinbacteria: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:amodelPalindromeDNAsequences

OligoUsStem-loop(hairpin)structureTranscriptionterminatorofE.coli

AddgeneModel

foranr-dependent terminator9.TranscriptionisahighlyregulatedprocessTranscriptionisthefirststepinthecomplicatedandenergy-expensivepathwayleadingtoproteinsynthesis,anidealtargetforregulatinggeneexpression.TheRNApolymerasebindstoeachpromoterinverydifferentefficiency.ProteinfactorsbindingtoDNAsequencescloseordistanttothepromoterscanpromote(activator)orrepress(repressor)thesynthesisofcertainRNAmolecules.10.ThreekindsofRNApolymerases(I,II,andIII)havebeenrevealedformakingRNAsinthenuclearsofeukaryoticcellsEachisresponsibleforthetranscriptionofacertaingroupsofgenes:rRNA,mRNAortRNAgenes.Theenzymesareoftenidentifiedbyexaminingtheirsensitivitytowardsa-amanitin(fromatoxicmushroom).EukaryoticRNApolymerases11.RNApolymeraseII(PolII)bindstopromotersofthousandsofprotein-codinggenesManyPolIIpromoterscontainaTATAAAsequence(calledaTATAbox)at-30positionandaninitiatorsequence(Inr)at+1position.Thepreinitiationcomplex(includingPolII)isbelievedtoassembleattheTATAbox,withDNAunwoundattheInrsequence.However,manyPolIIpromoterslackaTATAorInrorbothsequences!Generalfeaturesofpromotersforprotein-codinggenesinhighereukaryotesGCboxCAATbox12.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.TBPDNAAproposedmodelforPolII-catalyzedmRNAsynthesis13.TheactionofRNApolymerasescanbespecificallyinhibitedThree-ring-containing,planarantibioticmoleculeslikeactinomycinDintercalatesbetweentwosuccessiveGCbasepairsinduplexDNA,preventingRNApolymerases(alltypes)tomovealongthetemplate(thustheelongationofRNAsynthesis).Rifampicin

(anantibiotic)bindstothebsubunitofbacterialRNApolymerases,preventingtheinitiationofRNAsynthesis.a-amanitin

blockseukaryoticmRNAsynthesisbybindingtoRNApolymeraseII.14.RNAmoleculesareoftenfurtherprocessedafterbeingsynthesizedontheDNAtemplateTheprimarytranscriptsofeukaryoticmRNAsareoftencappedatthe5`end,splicedinthemiddle(intronsremovedandexonslinked),polyadenylatedatthe3`end.TheprimarytranscriptsofbothprokaryoticandeukaryotictRNAsarecleavedfrombothends,splicedinsomecases,andmodifiedformanyofthebasesandsugars.15.AneukaryoticmRNAprecursoracquirea5`capshortlyaftertranscriptioninitiatesAGMPcomponent(fromaGTP)isjoinedtothe5`endofthemRNAinanovel5`,5`-triphosphatelinkage.Theguaninebaseisthen

methylatedattheN-7.The2`-OHgroupsofthe1stand2ndnucleotidesadjacenttothe7-methylguaninecapmayalsobemethylatedincertainorganisms.ThemethylgroupsaretransferredfromS-adenosylhomocysteine.A5`capisaddedtoeukaryoticmRNAsBeforetranscriptionendsThe5`capfoundontheeukaryoticmRNAs16.MosteukaryoticmRNAshaveapoly(A)tailatthe3`endThetailconsistsof80to250adenylateresidues.ThemRNAprecursorsareextendedbeyondthesitewherepoly(A)tailistobeadded.AnAAUAAAsequencewasfoundtobepresentinallmRNAsandmarks(togetherwithothersignalsatthe3`end)thesiteforcleavageandpoly(A)tailaddition(11to30nucleotidesonthe3`endoftheAAUAAAsequence).Thespecificendonucleaseandpolyadenylatepolymerase,andotherproteinsprobablyexistasamultiproteincomplextocatalyzethisevent.Apoly(A)tailisusuallyaddedatthe3`endofanmRNAmoleculeviaaprocessingstep.17.EMstudiesofmRNA-DNAhybridsrevealedthediscontinuityofeukaryoticgenesEachgenewasfoundtobeacontinuousfragmentofDNAinthebacterialgenome.ButBergetandSharp(1977)observedsingle-strandedDNAloopswhenexaminingadenovirus

mRNA-DNAhybridsbyelectronmicroscopy.Suchsingle-strandedDNAloopswaswidelyobservedwhenexaminingsuchRNA-DNAhybrids.IntronsequenceswereproposedtobepresentonthetemplateDNAsequences,whichareremovedduringRNAprocessing,withexonslinkedtogetherprecisely.Almostallgenesinvertebratescontainintrons(buthistonegenesdoesnot).Manygenesincertainyeastsdonotcontainintrons.Intronsarealsofoundinafewbacterialandarchaebacterialgenes(butfarlesscommonthanineukaryoticcells).EMstudiesofmRNA-DNAhybridsforthechickenovalbumingene(theR-loopingtechnique)18.FourclassesofintronshavebeenrevealedhavingdifferentsplicingmechanismsGroupIintronsarefoundinsomenuclear,mitochondrialandchloroplastgenesencodingrRNAs,mRNAs,andtRNAs.GroupIIintronsareoftenfoundingenesencodingmRNAsinmitochondrialandchloroplastDNAoffungi,algae,andplants.GroupIIIintrons(thelargestgroup)arefoundingenesencodingeukaryoticnuclearmRNAs.GroupIVintronsarefoundingenesencodingthetRNAsinthenucleargenomicDNAofeukaryotes19.GroupIintronsareself-splicinganduseaguaninenucleosideornucleotideasthecofactorTheintronpresentintherRNAprecursorofTetrahymenawasfoundtoberemovedbyitselfwithoutusinganyproteins(ThomasCech,1982).Theintronisremovedandthetwoexonspreciselylinkedviatwonucleophilictransesterificationreactions(withtwo3`-OHgroupactasthenucleophiles).GroupIintronsareremovedbyself-splicingviatwonucleophilictransesterificationreactions.Thepredictedsecondarystructureoftheself-splicingrRNAintronofTetrahymenaTheinternalguidesequence5`splicesite3`splicesite20.GroupIIintronsalsoundergoself-splicingusingformingalariat-likeintermediate

Butthe2`-OHgroupofanadenylateresiduewithininremovingintronplayedtheroleofthe3`-OHgroupoftheguaninenucleosideornucleotideingroupIintronself-splicing.GroupIIintronsareremovedviaself-splicingwithanadenylateresidueoftheremovingintronactsasthenucleophile,forminganlariate-likeIntermediate.21.TypeIIIintronsarefoundinthenuclearmRNAprimarytranscriptsandhavethelargestnumbersThesplicingexon-intronjunctions,determinedbycomparingthesequencesofthegenomicDNAwiththatofthe

cDNApreparedfromthecorrespondingmRNA,inmRNAprecursorsarespecifiedbysequencesatthetwoendsoftheintrons:beginwith

GUandendwithAG.TypeIIIintronsareremovedviaaverysimilarwayasthatoftypeIIintronsexceptbeinghelpedbyseveralhighlyconservedsmallnuclearribonucleoproteins(snRNPs),eachcontainingaclassofU-richsmallnuclearRNAs(snRNAs).TypeIIIintrons,foundonnuclearmRNAprimarytranscripts,areremovedviathespliceosomes22.groupIVintronsarefoundintRNAprecursorsandareremovedbyendonucleaseandRNAligaseThesplicingendonucleasefirstcleavesthephosphodiesterbondsatbothendsoftheintron.ATPisneededfortheRNAligaseactivitytojointhetwoexons.ThejoiningreactionissimilartotheDNAligase-catalyzedreaction.ThemechanismofcleavinggroupIVintronsisdifferentfromthatofgroupI,II,andIIIintrons,allincludingtwotransesterificationreactions.GroupIVintronsaresplicedviatheactionofspecificendonucleaseandRNAligase.RNAligase23.AlternativeproteinsmaybeproducedfromonesinglegeneviadifferentialRNAprocessingThemultipletranscriptsproducedfromsuchagenemayhavemorethanonesiteforcleavageandpolyadenylation(asforimmunoglobulinheavychains),alternativesplicing(asforthemyosinheavychainsinfruitflies),orboth(asforthecalcitoningeneinrats).Indifferentcellsoratdifferentstagesofdevelopment,thetranscriptmaybeprocesseddifferentlytoproducedifferentgeneproducts(proteins).MultiplemRNAs(thuspolypeptidechains)canbeproducedviadifferentialRNAprocessing.24.ThedifferentrRNAmoleculesofbothprokaryotesandeukaryotesaregeneratedfromsinglepre-rRNAsThe16S,23Sand5SrRNAs(togetherwithcertaintRNAs)inbacteriaareallgeneratedfromasingle30Spre-rRNA(about6.5kb,transcribedbyRNApolymeraseI).Therearesevenpre-rRNAgenesintheE.coli

genome(eachencodingadifferenttRNA).The18S,28Sand5.8SrRNAsineukaryotesaregeneratedfromasingle40Spre-rRNA(~14kb).The5SrRNAineukaryoticcellsisgeneratedseparately(transcribedbyRNApolymeraseIII).AlltherRNAsarederivedfromasingleprecursorinprokaryoticcells.The18S,5.8S,and28SrRNAsineukaryoticcellsarederivedfromonepre-rRNAmolecule(theprocessingneedssmallnucleolarRNA-containingproteins).25.PrimarytRNAtranscriptsundergoaseriesofposttranscriptionalprocessingTheextrasequencesatthe5`and3`endsareremovedbyRNasePandRNaseDrespectively.TheRNAinRNasePiscatalytic(Altman,1983)TypeIVintrons

areoccasionallypresentinpre-tRNAsineukaryoticcells.TheCCAsequenceisgeneratedatthe3`endbytheactionoftRNAnucleotidyltransferase(havingthreeactivesitesforthethreeribonucleotidesadded).SomeofthebasesintRNAmoleculesaremodifiedbymethylation,deamination,reductionandothers.TheprocessingoftheprimarytRNA

transcriptsincluderemovalofthe5`and3`ends,additionoftheCCAsequenceatthe3`end,modificationofmanybases,andsplicingofintrons(ineukaryoticcells).SometypicalmodificiedbasesfoundInmaturetRNAmolecules.26.

MoreRNAmolecules(ribozymes)werefoundtobecatalyticCatalyticRNAmoleculeswerealsofoundinthevirusoidRNA(calledhammerheadribozymes).RNAsinthespliceosomes(theU-richRNAs)andribosomesarealsobelievedtobecatalytic.Aspecific3-Dstructureisrequiredforribozymestobecatalytic.Ribozymesoftenorienttheirsubstratesviabasepairing.Theexcisedintron(414nucleotides)ofthepre-rRNAof

TetrahymenaisfurtherprocessedtoaRNAfragmentof395nucleotidesnamedasL-19IVS;(interveningsequencelacking19nucleotides)Aportionoftheinternalguidesequenceremainsatthe5`endofL-19IVSandtheguanosinebindingsiteisstillintact.Dr.CechreasonedthatL-19IVSmightactonexternalsubstrates.L-19IVSisabletocatalyzethelengtheningofsomeoligonucleotides,likea(C)5oligomer,attheexpenseofothers(beingbothanucleaseand

polymerase).L-19IVS

functionsasarealcatalystinthetesttubeIncubationtime(minutes)LabeledsubstrateRNANucleaseactivityRNApolymeraseactivityTheM1RNAinribonucleasePiscatalyticTheintroninthepre-rRNAofTetrahemenaisself-spliced27.ThecellularmRNAsaredegradedatdifferentratesThelevelofaproteininacellisdeterminedtosomeextentbythelevelofitsmRNA,whichdependsonabalanceoftheratesonitssynthesisanddegradation.ThehalfoflivesofdifferentmRNAmoleculesvarygreatly,fromsecondstomanycellgenerations.3`hairpinandpoly(A)tailshavebeenshowntoincreasehalvelivesofmRNAs,butmultiple,sometimesoverlappingAUUUAsequenceshavebeenshowntodecreasehalvelives.

The5`3`exoribonucleaseisprobablythemajordegradingenzymeformRNAs.Thepolynucleotidephosphorylase

maybeanotherenzymedegradingmRNAs.PolynucleotidephosphorylasewasusedtosynthesizeRNAforthefirsttimeinthetesttube(SeveroOchoasharedtheNobelPrizewithArthurKornbergin1959forthisdiscovery).(NMP)n+1+Pi (NMP)n+NDPThisenzymewasusedtosynthesizeRNApolymersofdifferentsequencesandfrequenciesofbasesfortheelucidationofthegeneticcodes.AveragehalflivesofmRNAmolecules

Bacteria 1.5minutes

Vertebrates 3hours

28.ReversetranscriptasescatalyzetheproductionofDNAfromRNATheexistenceofthisenzymeinretroviruses(RNAviruses)waspredictedbyHowardTeminin1962,andprovedbyTeminandDavidBaltimorein1970.Thisenzymecatalyzesthreereactions:RNA-directedDNAsynthesisusingtRNAsasprimers;DegradationoftheRNAtemplate;DNA-directedDNAsynthesis;Theenzymehasno3`5`proofreadingexonuclease

activity,thusgeneratinghighrateofmutations.ThisenzymeiswidelyusedtosynthesizecomplementaryDNAs(cDNAs)frommRNAs.ReversetranscriptasescatalyzesthesythesisofDNAfromRNAtemplate.29.Telomerasecatalyzesthesynthesisoftherepeatingtelomeresequences(TxGy)usinganinternalRNAtemplateTelomeresconsistofafewtoalargenumberoftandemcopiesofashortoligonucleotidesequencethatarelocatedatthetwoendsofthelinearchromosomalDNAs,havinga3`singlestrandextension(ontheTGstrand).

Telomeraseactstopreventthechromosomalendsfrombecomingshortenedaftereachreplication(theendpartofthelaggingstrandcannotbeduplicated).Telomeraseisactuallyareversetranscriptase,butusesashortsegmentofaninternalRNAmolecule(~150nucleotides)asthetemplatetoextendtheend.TheCyAx

strand(thelaggingstrand)isbelievedtobesynthesizedbyaDNApolymeraseusingaRNAprimer.Theendsofalinearchromosomeisoftenprotectedbybindingtospecificproteins,formingaTloopstructureinhighereukaryotes,wherethesingle-strandedDNAissequestered.Thelengthofthetelomereseemstobeinverselyrelatedtothelifespanofcellsandindividuals(shortensasoneages).ProblemposedinthereplicationoflinearDNA:theendofonedaughterstrandwillbeshortenedaftereachroundofreplication.The“inchworm”(尺蠖）modelfortelomeraseactionTheTloopobservedatoneendofamammalianchromosome.30.SomeviralRNAsarereplicatedbyRNA-directedRNApolymeraseTheRNAgenomesofsomeviruses(havingbacteria,animalsorplantsastheirhosts)arereplicatedusingRNA-directedRNApolymerases(orRNAreplicases).TheRNAreplicasefrombacteriophage-infectedE.coli

cellsconsistsofsubunitsencodedbothbythevirusesandthehostgenome.Theyhavefeaturessimilarto