Translation: DNA to mRNA to Protein | Learn Science at Scitable

In this situation, translation begins at the 5' end of the mRNA while the 3' end is still ... the order in which this occurs is unique to prokaryotic cells. Thispagehasbeenarchivedandisnolongerupdated   Translation:DNAtomRNAtoProtein By: SuzanneClancy,Ph.D. & WilliamBrown,Ph.D. (WriteScienceRight) © 2008 NatureEducation  Citation: Clancy, S. & Brown, W. (2008) Translation:DNAtomRNAtoProtein. NatureEducation 1(1):101 HowdoesthecellconvertDNAintoworkingproteins?Theprocessoftranslationcanbeseenasthedecodingofinstructionsformakingproteins,involvingmRNAintranscriptionaswellastRNA. Aa Aa Aa  ThegenesinDNAencodeproteinmolecules,whicharethe"workhorses"ofthecell,carryingoutallthefunctionsnecessaryforlife.Forexample,enzymes,includingthosethatmetabolizenutrientsandsynthesizenewcellularconstituents,aswellasDNApolymerasesandotherenzymesthatmakecopiesofDNAduringcelldivision,areallproteins.Inthesimplestsense,expressingagenemeansmanufacturingitscorrespondingprotein,andthismultilayeredprocesshastwomajorsteps.Inthefirststep,theinformationinDNAistransferredtoamessengerRNA(mRNA)moleculebywayofaprocesscalledtranscription.Duringtranscription,theDNAofageneservesasatemplateforcomplementarybase-pairing,andanenzymecalledRNApolymeraseIIcatalyzestheformationofapre-mRNAmolecule,whichisthenprocessedtoformmaturemRNA(Figure1).TheresultingmRNAisasingle-strandedcopyofthegene,whichnextmustbetranslatedintoaproteinmolecule.Figure1: Ageneisexpressedthroughtheprocessesoftranscriptionandtranslation.Duringtranscription,theenzymeRNApolymerase(green)usesDNAasatemplatetoproduceapre-mRNAtranscript(pink).Thepre-mRNAisprocessedtoformamaturemRNAmoleculethatcanbetranslatedtobuildtheproteinmolecule(polypeptide)encodedbytheoriginalgene.©2013NatureEducationAllrightsreserved.FigureDetailDuringtranslation,whichisthesecondmajorstepingeneexpression,themRNAis"read"accordingtothegeneticcode,whichrelatestheDNAsequencetotheaminoacidsequenceinproteins(Figure2).EachgroupofthreebasesinmRNAconstitutesacodon,andeachcodonspecifiesaparticularaminoacid(hence,itisatripletcode).ThemRNAsequenceisthususedasatemplatetoassemble—inorder—thechainofaminoacidsthatformaprotein.Figure2: TheaminoacidsspecifiedbyeachmRNAcodon.Multiplecodonscancodeforthesameaminoacid.Thecodonsarewritten5'to3',astheyappearinthemRNA.AUGisaninitiationcodon;UAA,UAG,andUGAaretermination(stop)codons.©2014NatureEducationAllrightsreserved.FigureDetailButwheredoestranslationtakeplacewithinacell?Whatindividualsubstepsareapartofthisprocess?Anddoestranslationdifferbetweenprokaryotesandeukaryotes?Theanswerstoquestionssuchastheserevealagreatdealabouttheessentialsimilaritiesbetweenallspecies.WhereTranslationOccursWithinallcells,thetranslationmachineryresideswithinaspecializedorganellecalledtheribosome.Ineukaryotes,maturemRNAmoleculesmustleavethenucleusandtraveltothecytoplasm,wheretheribosomesarelocated.Ontheotherhand,inprokaryoticorganisms,ribosomescanattachtomRNAwhileitisstillbeingtranscribed.Inthissituation,translationbeginsatthe5'endofthemRNAwhilethe3'endisstillattachedtoDNA.Inalltypesofcells,theribosomeiscomposedoftwosubunits:thelarge(50S)subunitandthesmall(30S)subunit(S,forsvedbergunit,isameasureofsedimentationvelocityand,therefore,mass).Eachsubunitexistsseparatelyinthecytoplasm,butthetwojointogetheronthemRNAmolecule.TheribosomalsubunitscontainproteinsandspecializedRNAmolecules—specifically,ribosomalRNA(rRNA)andtransferRNA(tRNA).ThetRNAmoleculesareadaptormolecules—theyhaveoneendthatcanreadthetripletcodeinthemRNAthroughcomplementarybase-pairing,andanotherendthatattachestoaspecificaminoacid(Chapevilleetal.,1962;Grunbergeretal.,1969).TheideathattRNAwasanadaptormoleculewasfirstproposedbyFrancisCrick,co-discovererofDNAstructure,whodidmuchofthekeyworkindecipheringthegeneticcode(Crick,1958).Withintheribosome,themRNAandaminoacyl-tRNAcomplexesareheldtogetherclosely,whichfacilitatesbase-pairing.TherRNAcatalyzestheattachmentofeachnewaminoacidtothegrowingchain.TheBeginningofmRNAIsNotTranslatedInterestingly,notallregionsofanmRNAmoleculecorrespondtoparticularaminoacids.Inparticular,thereisanareanearthe5'endofthemoleculethatisknownastheuntranslatedregion(UTR)orleadersequence.ThisportionofmRNAislocatedbetweenthefirstnucleotidethatistranscribedandthestartcodon(AUG)ofthecodingregion,anditdoesnotaffectthesequenceofaminoacidsinaprotein(Figure3).So,whatisthepurposeoftheUTR?Itturnsoutthattheleadersequenceisimportantbecauseitcontainsaribosome-bindingsite.Inbacteria,thissiteisknownastheShine-Dalgarnobox(AGGAGG),afterscientistsJohnShineandLynnDalgarno,whofirstcharacterizedit.AsimilarsiteinvertebrateswascharacterizedbyMarilynKozakandisthusknownastheKozakbox.InbacterialmRNA,the5'UTRisnormallyshort;inhumanmRNA,themedianlengthofthe5'UTRisabout170nucleotides.Iftheleaderislong,itmaycontainregulatorysequences,includingbindingsitesforproteins,thatcanaffectthestabilityofthemRNAortheefficiencyofitstranslation.Figure3: ADNAtranscriptionunit.ADNAtranscriptionunitiscomposed,fromits3'to5'end,ofanRNA-codingregion(pinkrectangle)flankedbyapromoterregion(greenrectangle)andaterminatorregion(blackrectangle).Regionstotheleft,ormovingtowardsthe3'end,ofthetranscriptionstartsiteareconsidered\"upstream;\"regionstotheright,ormovingtowardsthe5'end,ofthetranscriptionstartsiteareconsidered\"downstream.\"©2014NatureEducationAdaptedfromPierce,Benjamin.Genetics:AConceptualApproach,2nded.Allrightsreserved.TranslationBeginsAftertheAssemblyofaComplexStructureThetranslationofmRNAbeginswiththeformationofacomplexonthemRNA(Figure4).First,threeinitiationfactorproteins(knownasIF1,IF2,andIF3)bindtothesmallsubunitoftheribosome.Thispreinitiationcomplexandamethionine-carryingtRNAthenbindtothemRNA,neartheAUGstartcodon,formingtheinitiationcomplex.Figure4: Thetranslationinitiationcomplex.Whentranslationbegins,thesmallsubunitoftheribosomeandaninitiatortRNAmoleculeassembleonthemRNAtranscript.Thesmallsubunitoftheribosomehasthreebindingsites:anaminoacidsite(A),apolypeptidesite(P),andanexitsite(E).TheinitiatortRNAmoleculecarryingtheaminoacidmethioninebindstotheAUGstartcodonofthemRNAtranscriptattheribosome’sPsitewhereitwillbecomethefirstaminoacidincorporatedintothegrowingpolypeptidechain.Here,theinitiatortRNAmoleculeisshownbindingafterthesmallribosomalsubunithasassembledonthemRNA;theorderinwhichthisoccursisuniquetoprokaryoticcells.Ineukaryotes,thefreeinitiatortRNAfirstbindsthesmallribosomalsubunittoformacomplex.ThecomplexthenbindsthemRNAtranscript,sothatthetRNAandthesmallribosomalsubunitbindthemRNAsimultaneously.©2013NatureEducationAllrightsreserved.FigureDetailAlthoughmethionine(Met)isthefirstaminoacidincorporatedintoanynewprotein,itisnotalwaysthefirstaminoacidinmatureproteins—inmanyproteins,methionineisremovedaftertranslation.Infact,ifalargenumberofproteinsaresequencedandcomparedwiththeirknowngenesequences,methionine(orformylmethionine)occursattheN-terminusofallofthem.However,notallaminoacidsareequallylikelytooccursecondinthechain,andthesecondaminoacidinfluenceswhethertheinitialmethionineisenzymaticallyremoved.Forexample,manyproteinsbeginwithmethioninefollowedbyalanine.Inbothprokaryotesandeukaryotes,theseproteinshavethemethionineremoved,sothatalaninebecomestheN-terminalaminoacid(Table1).However,ifthesecondaminoacidislysine,whichisalsofrequentlythecase,methionineisnotremoved(atleastinthesampleproteinsthathavebeenstudiedthusfar).Theseproteinsthereforebeginwithmethioninefollowedbylysine(Flintaetal.,1986).Table1showstheN-terminalsequencesofproteinsinprokaryotesandeukaryotes,basedonasampleof170prokaryoticand120eukaryoticproteins(Flintaetal.,1986).Inthetable,Mrepresentsmethionine,Arepresentsalanine,Krepresentslysine,Srepresentsserine,andTrepresentsthreonine.Table1:N-TerminalSequencesofProteinsN-TerminalSequencePercentofProkaryoticProteinswithThisSequencePercentofEukaryoticProteinswithThisSequenceMA*28.24%19.17%MK**10.59%2.50%MS*9.41%11.67%MT*7.65%6.67%*Methioninewasremovedinalloftheseproteins**MethioninewasnotremovedfromanyoftheseproteinsOncetheinitiationcomplexisformedonthemRNA,thelargeribosomalsubunitbindstothiscomplex,whichcausesthereleaseofIFs(initiationfactors).ThelargesubunitoftheribosomehasthreesitesatwhichtRNAmoleculescanbind.TheA(aminoacid)siteisthelocationatwhichtheaminoacyl-tRNAanticodonbasepairsupwiththemRNAcodon,ensuringthatcorrectaminoacidisaddedtothegrowingpolypeptidechain.TheP(polypeptide)siteisthelocationatwhichtheaminoacidistransferredfromitstRNAtothegrowingpolypeptidechain.Finally,theE(exit)siteisthelocationatwhichthe"empty"tRNAsitsbeforebeingreleasedbackintothecytoplasmtobindanotheraminoacidandrepeattheprocess.TheinitiatormethioninetRNAistheonlyaminoacyl-tRNAthatcanbindinthePsiteoftheribosome,andtheAsiteisalignedwiththesecondmRNAcodon.Theribosomeisthusreadytobindthesecondaminoacyl-tRNAattheAsite,whichwillbejoinedtotheinitiatormethioninebythefirstpeptidebond(Figure5).Figure5: Thelargeribosomalsubunitbindstothesmallribosomalsubunittocompletetheinitiationcomplex.TheinitiatortRNAmolecule,carryingthemethionineaminoacidthatwillserveasthefirstaminoacidofthepolypeptidechain,isboundtothePsiteontheribosome.TheAsiteisalignedwiththenextcodon,whichwillbeboundbytheanticodonofthenextincomingtRNA.©2013NatureEducationAllrightsreserved.TheElongationPhaseFigure6FigureDetailThenextphaseintranslationisknownastheelongationphase(Figure6).First,theribosomemovesalongthemRNAinthe5'-to-3'direction,whichrequirestheelongationfactorG,inaprocesscalledtranslocation.ThetRNAthatcorrespondstothesecondcodoncanthenbindtotheAsite,astepthatrequireselongationfactors(inE.coli,thesearecalledEF-TuandEF-Ts),aswellasguanosinetriphosphate(GTP)asanenergysourcefortheprocess.UponbindingofthetRNA-aminoacidcomplexintheAsite,GTPiscleavedtoformguanosinediphosphate(GDP),thenreleasedalongwithEF-TutoberecycledbyEF-Tsforthenextround.Next,peptidebondsbetweenthenow-adjacentfirstandsecondaminoacidsareformedthroughapeptidyltransferaseactivity.Formanyyears,itwasthoughtthatanenzymecatalyzedthisstep,butrecentevidenceindicatesthatthetransferaseactivityisacatalyticfunctionofrRNA(Pierce,2000).Afterthepeptidebondisformed,theribosomeshifts,ortranslocates,again,thuscausingthetRNAtooccupytheEsite.ThetRNAisthenreleasedtothecytoplasmtopickupanotheraminoacid.Inaddition,theAsiteisnowemptyandreadytoreceivethetRNAforthenextcodon.ThisprocessisrepeateduntilallthecodonsinthemRNAhavebeenreadbytRNAmolecules,andtheaminoacidsattachedtothetRNAshavebeenlinkedtogetherinthegrowingpolypeptidechainintheappropriateorder.Atthispoint,translationmustbeterminated,andthenascentproteinmustbereleasedfromthemRNAandribosome.TerminationofTranslationTherearethreeterminationcodonsthatareemployedattheendofaprotein-codingsequenceinmRNA:UAA,UAG,andUGA.NotRNAsrecognizethesecodons.Thus,intheplaceofthesetRNAs,oneofseveralproteins,calledreleasefactors,bindsandfacilitatesreleaseofthemRNAfromtheribosomeandsubsequentdissociationoftheribosome.ComparingEukaryoticandProkaryoticTranslationThetranslationprocessisverysimilarinprokaryotesandeukaryotes.Althoughdifferentelongation,initiation,andterminationfactorsareused,thegeneticcodeisgenerallyidentical.Aspreviouslynoted,inbacteria,transcriptionandtranslationtakeplacesimultaneously,andmRNAsarerelativelyshort-lived.Ineukaryotes,however,mRNAshavehighlyvariablehalf-lives,aresubjecttomodifications,andmustexitthenucleustobetranslated;thesemultiplestepsofferadditionalopportunitiestoregulatelevelsofproteinproduction,andtherebyfine-tunegeneexpression.ReferencesandRecommendedReadingChapeville,F.,etal.Ontheroleofsolubleribonucleicacidincodingforaminoacids.ProceedingsoftheNationalAcademyofSciences48,1086–1092(1962)Crick,F.Onproteinsynthesis.SymposiaoftheSocietyforExperimentalBiology12,138–163(1958)Flinta,C.,etal.SequencedeterminantsofN-terminalproteinprocessing.EuropeanJournalofBiochemistry154,193–196(1986)Grunberger,D.,etal.Codonrecognitionbyenzymaticallymischargedvalinetransferribonucleicacid.Science166,1635–1637(1969)doi:10.1126/science.166.3913.1635Kozak,M.PointmutationsclosetotheAUGinitiatorcodonaffecttheefficiencyoftranslationofratpreproinsulininvivo.Nature308,241–246(1984)doi:10.1038308241a0(linktoarticle)---.PointmutationsdefineasequenceflankingtheAUGinitiatorcodonthatmodulatestranslationbyeukaryoticribosomes.Cell44,283–292(1986)---.Ananalysisof5'-noncodingsequencesfrom699vertebratemessengerRNAs.NucleicAcidsResearch15,8125–8148(1987)Pierce,B.A.Genetics:Aconceptualapproach(NewYork,Freeman,2000)Shine,J.,&Dalgarno,L.Determinantofcistronspecificityinbacterialribosomes.Nature254,34–38(1975)doi:10.1038/254034a0(linktoarticle) 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