分子生物学Chapter 10 Protein Synthesis.docx
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分子生物学Chapter 10 Protein Synthesis.docx
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分子生物学Chapter10ProteinSynthesis
Chapter10ProteinSynthesis
Introduction
TranslationisthewholeprocessbywhichthebasesequenceofanmRNAisusedtoorderandtojointheaminoacidsinaprotein.
1.THEGENETICCODE
vGeneticcodeisatripletcodeinmRNA(Threenucleotidesencodeoneaminoacid)
vThetripletcodonsarenon-overlappingandcomma-less.
Geneticcodeisdegenerate:
Only20aminoacidsareencodedby4nucleotidesintripletcodons(43=64ofaminoacidscouldpotentiallybeencoded).Therefore,morethanonetripletsareusedtospecifyaaminoacids,andthegeneticcodesaresaidtobedegenerate,ortohaveredundancy.
ORFs
Openreadingframes(ORFs)aresuspectedcodingregionsstartingwithATGandendwithUAG(amber),TAAochre)orUGA(opal)identifiedbycomputer.
WhentheORFisknowntoencodeacertainprotein,itisusuallyreferredasacodingregion.
Overlappinggenes
vGenerallytheseoccurwherethegenomesizeissmall(virusesinmostcases)andthereisaneedforgreaterinformationstoragedensity.
vMorethanonestartcodonsinaDNAsequenceareusedfortranslatingdifferentproteins.
vAwaytomaximizethecodingcapabilityofagivenDNAsequence.
vExampleofoverlappinggenes:
vPhageX174with5386bp(maximal200kDa),infact,itencodes11proteinsof262kDaintotal)
Chapter10ProteinSynthesis
2.tRNA
vtRNAaretheadaptormoleculesthatdeliveraminoacidstotheribosomeanddecodetheinformationinmRNA.
v2.1.tRNAStructure:
v1°structure:
small,75nucleotides,manymodifiedbases:
Di-hydroU;Pseudo-U:
Y;modifiedG's;Thymine...
v
tRNAsecondarystructure
vThecloverleafstructureisacommonsecondarystructuralrepresentationoftRNAmoleculeswhichshowsthebaseparingofvariousregionstoformfourstems(arms)andthreeloops.
v2°structure:
cloverleafof4majorstem-loops,1minorstem-loop
namesofarms:
D-arm(dihydroarm),AminoAcylorAcceptorarm,T(pseudoU)Carm,Extraarm(Type1:
short;Type2:
long),Anticodonarm
vThe5’-and3’-endarelargelybase-pairedtoformtheaminoacidacceptorstemwhichhasnoloop.
Composedof3or4bpstemandaloopcalledtheD-loop(DHU-loop)usuallycontainingthemodifiedbasedihydrouracil.
Consistingofa5bpstemanda7residuesloopinwhichtherearethreeadjacentnucleosidescalledtheanticodonwhicharecomplementarytothecodonsequence(atripletinthemRNA)thatthetRNArecognize.
Variablearm:
3to21residuesandmayformastemofupto7bp.
T-armiscomposedofa5bpstemendinginaloopcontainingtheinvariantresiduesGTC.
v3°structure:
L-shape,withformationofadditionalH-bondinteractions,someofwhicharenonstandardW-Cbaseinteractions.
2.2.tRNAFunction:
bringaminoacidstotheribosomesforproteinsynthesisviacovalentlinkageofaminoacidtothetRNA:
aminoacyl-tRNAspecies
FunctionofeachtRNAarm:
v1)AminoAcylorAcceptorarm:
covalentlybindtheaminoacid;interactwithaminoacyltRNAsynthetases;providesubstrateforpeptidyltransferaseinthelargesubunitribosome.
2)D-arm(Dihydroarm):
importantin3DstructureoftRNA;interactswithlargesubunitribosomeinPandAsites
2.3.Aminoacyl-tRNASynthetases
vEachtRNAmoleculeisrecognizedbyoneandonlyoneofthe20aminoacyl-tRNAsynthetases.
Two-stepCatalyticReaction
v1)ATP+aa-->aa-AMP+PPi,andPPi-->Pi+Pi
vNH3+
v|O-
vwhereaa-AMPis:
R-C-CO-O-P-O-Ribose-Adenine
vHO
v2)aa-AMP+tRNA-->aa-tRNA+AMP
TwoclassesofAminoacyl-tRNASynthetases:
bindtoopposite"sides"ofthetRNA
vClassI:
bindsminorgrovesofacceptorandanticodonstems;ATPbindingdomainanucleotidebindingdomainatN-termendofenzyme;anticodonarmbindingdomainatCtermend;synthetaseattachesaatothe2'-OHoftheriboseofthe3terminalnucleotide(theAofthe-CCA)ofthetRNA...
vClassII:
bindsmajorgrovesofacceptorandanticodonstems;anticodonarmboundinmajorgroovebyN-termdomain;ATPbindingdomainmoreC-term;synthetaseattachesaatothe3'-OHoftheriboseofthe3terminalnucleotide(theAofthe-CCA)ofthetRNA..
tRNA"Identity"Elements
vMostorganismsencodeclosetoOneSynthetaseperaminoacid
vProblem:
Ifthereareroughly1tRNAspeciespercodonbutonlyoneSynthetaseperaminoacid,thenaSynthetaseforanaminoacidwhichhasmorethanonecodonmustbeabletospecificallyrecognizeandbindeachofthetRNAspeciesforeachofthesecodons.
vAnswer(inpart...muchisstillunknown):
tRNAspeciesthatencodeagivenaminoacidcontain
vIdentityElements.ThesearenucleotidesfoundincommonbetweenthesetRNAspecies,butwhichare,atleastinpart,differentinallothertRNAspecies.
IdentityElementNucleotides
vThesearefoundprimarilyintwolocations:
1)theAnticodon
2)the5'-terminalnucleotidesandthe3'-terminalnucleotidesadjacenttothe-CCAresidues
SomearealsofoundintheD-loopstem-loop
TherecognitionofatRNAmoleculebyitsaminoacyl-tRNAsynthetase.
2.4.Wobblehypothesis:
codon-anticodonrecognition
vAsstudiesontRNAproceeded,30–40differenttRNAswereidentifiedinbacterialcellsandasmanyas50–100inanimalandplantcells.ThusthenumberoftRNAsinmostcellsismorethanthenumberofaminoacidsfoundinproteins(20)andalsodiffersfromthenumberofcodonsinthegeneticcode(61).
vConsequently,manyaminoacidshavemorethanonetRNAtowhichtheycanattach(explaininghowtherecanbemoretRNAsthanaminoacids);inaddition,manytRNAscanattachtomorethanonecodon(explaininghowtherecanbemorecodonsthantRNAs).Asnotedpreviously,mostaminoacidsareencodedbymorethanonecodon,requiringsometRNAstorecognizemorethanonecodon.
vConsequently,manyaminoacidshavemorethanonetRNAtowhichtheycanattach(explaininghowtherecanbemoretRNAsthanaminoacids);inaddition,manytRNAscanattachtomorethanonecodon(explaininghowtherecanbemorecodonsthantRNAs).Asnotedpreviously,mostaminoacidsareencodedbymorethanonecodon,requiringsometRNAstorecognizemorethanonecodon.
vDegeneracyorRedundancyinthegeneticcodeoccursprimarilyinthe3rdpositionofthecodon.Toaccountforthis,CrickproposedtheWobbleHypothesis.Thishypothesisstatesthatbasepairingofbasesatthe3rdpositionofthecodonandthe1stpositionoftheanticodoncanbenonstandarddueto"wobble"ofthenucleotideatthe1stpositionoftheanticodon.SuchwobbleisduetothecurvatureoftheanticodonintheloopoftheanticodonarmofthetRNA.
vThefirstandsecondbasesinanmRNAcodonformWatson-Crickbasepairswiththethirdandsecondbases,respectively,ofatRNAanticodon.
Thenonstandard,wobblebasepairsU·G,C·I,A·I,andU·I.
2.5.SuppressortRNAs:
vExampleofSuppression:
asecondmutation(intheanticodonnucleotidesofatRNAgene)whichsuppressestheeffectsofafirstmutation(inthegenebeingtranslated).
vmutatedAntiCodons
vSuppressortRNAssuppressmutationspresentinanmRNAduringtranslationbyinsertingan"incorrect"aminoacidintothegrowingpolypeptidechain.
vTypesofsuchsuppression:
suppressionofnonsense,missense,frameshiftmutations
1)Nonsensesuppression:
amber,ochre,opalmutantsinthemRNA
vAmbermutation:
sensecodonmutatedtoUAG
vOchremutation:
sensecodonmutatedtoUAA
vOpalmutation:
sensecodonmutatedtoUGA
vEachofthesemutationsyieldsaStopCodoninthemiddleofagene,resultinginsynthesisofatruncated(shortened)polypeptidechain.Suchpolypeptidesarenearlyalwaysinactiveasenzymes.
vAsuppressortRNArecognizesthemutantstopcodon,andinsertsanaminoacidinthispositionofthepolypeptideduringtranslation.Theresultingnon-truncatedpolypeptidewillbeenzymaticallyactiveifitcantoleratetheaminoacidchangeatthisposition.
2)Missensesuppression:
vsimilartononsensesuppressionbutsuppressionofamissensemutation
3)Frameshiftsuppression:
vframeshiftmutantsarenotsuppressedbysuppressortRNAs.However,theycanbesuppressedbyslippageoftheribosomebyonenucleotide.Suchisusuallydonebymutantribosomes.Hence,thesuppressionisbyasecondmutationinaribosomalproteingene.
Chapter10ProteinSynthesis
3.Ribosomes
v3.1.RibosomeStructure:
Prokaryotes:
30Ssmallsubunit+50Slargesubunit->70Scompleteribosome
HigherEukaryotes:
40Ssmallsubunit+60Slargesubunit->80Scompleteribosome
Thegeneralstructureofribosomesinprokaryotesandeukaryotes.
3.2.RibosomeBindingSites
v3sitesfortRNA:
Asite(entrysite),Psite(aatransfer),Esite(exit)
mRNAsiteon30Sribosome
Peptidyltransferasesiteon50Sribosome
Chapter10ProteinSynthesis
4.InitiationofTranslationProcess
vDuringtheinitialstageofproteinsynthesisinallcells,aribosomeassembles,complexedwithamRNAandanactivatedinitiatortRNA,whichiscorrectlypositionedatthestartcodon.Becausethedetailsofinitiationandthemechanismforlocatingthetranslationstartsitedifferinbacteriaandeukaryotes,wediscussthetwosystemsseparately.
4.1.Prokaryotic
v4.1.1.mRNAhastworecognitionsites:
v1).TheShine-DelgarnoribosomebindingsiteAGGAGG,complementaryinsequencetoaCCUCCUsequenceon16SrRNAonthe30Sribosome
v2).ThetranslationStartCodon,usuallyAUG,sometimesGUG
4.1.2.fMet-tRNAf
vAspecificaminoacyl-tRNAusedinInitiation:
TheformylationreactionoccursaftertRNAfischargedwithMethionine
Thisreactionusesformyl-tetrahydrofolateasformyldonor.
vAdeformylaseremovestheFormy
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