分子生物学学习资料：MB answers 03

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AnswerstoWeaverendofchapterquestions

Chapter3IntroductiontoGeneFunction

Structureofanhelix.

Structureofa-pleatedsheet.

Thishelicalstructureisstabilizedbybondingbetweenadjacentsidechainsofaminoacids.

Thisstructureisstabilizedbyhydrogenbondingbetweenatomsintheadjacentpolypeptidechainbackbones.

Theprimarystructureofaproteinisthelinearorderofaminoacidsinthepolypeptidechainjoinedbycovalentpeptidebonds.Thesecondarystructurereferstotheformationofeither-helicesor-pleatedsheetsbythepeptidechain.-helicesarestabilizedbyhydrogenbondingbetweenthesidechainsoftheaminoacidswhereas-pleatedsheetsresultfrompackingofpolypeptidechainssidebysidejoinedbyhydrogenbondingbetweenparallelbackbonesofthechain.Thesetwotypesofsecondarystructurecanbelinkedbyturnsinthepolypeptidechain.Thetertiarystructureistheoverallthreedimensionalshapeofaproteinasitfoldsintoitsfunctionalconformation.Thisshapeisaresultofanumberoftypesofinteractionsbetweenfunctionalgroupsontheaminoacidsincluding,ionicbonding,hydrogenbonding,andhydrophobicinteractions.Althoughnon-covalentinteractionsplaythemostsignificantroleinstabilizingthetertiarystructureofaprotein,covalentbonds,primarilydisulphidebondsbetweencysteines,areoftenpresent.Thequaternarystructureofaproteinreferstotheassemblingoftwoormoreseparatepolypeptidechainstoformthefunctionalprotein.Thequaternarystructureofaproteinisstabilizedmainlybyhydrophobicinteractions.

ArchibaldGarrodisthephysicianwhowascreditedwithnotingthehereditarynatureofthedisease,Alcaptonuria.BeingfamiliarwithMendel’swork,heproposedthatasinglerecessivegenecausedthedisease.Healsohypothesizedthatthedisorderwascausedbyadefectinametabolicpathwayandthus,adefectiveenzyme.Puttingthesetwoobservationstogether,hemadetheconnectionbetweentheproteinencodingtheenzymeandtherecessivegene.Thissuggestedthatgenesencodeproteins.

BeadleandTatumusedtheexperimentalsystem,Neurosporacrassa,todemonstratetherelationshipbetweengenesandproteins.Neurosporaisahaploideukaryotethatcanreproducesexuallybyfusingtwohaploidnucleiwhichthenproducehaploidsporesbymeiosis.Inordertogrow,thefungusreliesuponaseriesofbiochemicalpathwaysthatcansynthesizeacompletesetofaminoacidsandvitaminsifthefungusisprovidedwithinorganicnitrogenandthesinglevitaminbiotin.ByirradiatingthesporeformingpartsofthefunguswithX-raysandperforminggeneticanalysisontheprogeny,theygeneratedmanypurestrainsofthefungus,eachofwhichcouldnotgrowonminimalmediabutwhichcouldgrowonamediumwhichcontainedalloftheaminoacidsandvitaminsthatthewild-typefunguscansynthesize.Theythensetaboutthelaborioustaskofdeterminingwhichstrainwasmutantinwhichbiochemicalpathway.Theyaccomplishedthisbydeterminingwhichstraincouldgrowwiththeadditionofaparticularvitamin,forexample,pantothenate.Astrainthatcouldgrowonlywhensupplementedwithaparticularvitaminhadamutationinthispathway.Theytestedtheabilityofvariousintermediatestoallowthefungustonowgrow(tocomplementthemutation)allowingthemtofurtherpinpointthespecificstepinthepathwaythatwasblocked.Ifastraincouldgrowwhenprovidedwithaparticularintermediate,theythenconcludedthatthemutationlayinasteppriortotheproductionofthatintermediate.Thustheybuiltupaseriesofmutants,eachwithaspecificenzymaticdefectinheritedasasinglegene.Thisdemonstratedtheconnectionbetweensinglegenesandthesinglepolypeptidescomprisingeachenzyme.

Thetwomainstepsingeneexpressionaretranscription,whichisthegenerationofanmRNAmoleculefromtheDNAtemplate,andtranslation,whichisthegenerationofapolypeptidechainasdeterminedbythelinearsequenceofcodonsinthemessengerRNA.

SeeFigure3.12.TheworkofJacobandcolleaguesdemonstratedtheexistenceofanRNAmolecule,messengerRNA,whichtransientlyassociateswiththeribosomeanddirectsproteinsynthesis.TheyprovidedevidencerefutingCrick’shypothesisthatthegeneticinformationiscarriedinribosomalRNAandisthereforeanintegralpartoftheribosome.TheyusedE.coliinfectedwithT2bacteriophageastheirexperimentalsystem.UponinfectionofE.coliwithphageT2,thereisamassiveshiftinproteinsynthesisasthebacteriabeginmakingphageproteins.Jacobetal.hypothesizedthat,ifproteinsynthesisisdirectedbyamessengerRNAmoleculenotpermanentlyassociatedwiththeribosomes,ashiftinproteinsynthesiswouldnotrequirereplacementofribosomeswithonescarryingtheinformationforthesynthesisofthenewphageproteins.Tolabelandtrackribosomesbeforeandafterphageinfection,theygrewE.coliinheavyisotopesofnitrogenandcarbonbeforeinfection.TheseE.colicultureswerethentransferredtomediacontainingthelightisotopesofcarbonandnitrogenand,atthesametime,theywereinfectedwithT2phageand32PwasaddedtolabelthenewlysynthesizedmRNA.Usingdensitygradientcentrifugationitwaspossibletoseparatethe“old”heavyribosomesfromthenewly-synthesizedlightribosomes.TheythendeterminedwhethertheRNAassociatedwitheachribosometypewaslabeledwith32Pandthereforenewlysynthesized.TheyshowedthatlabeledRNAwasassociatedwiththeribosomesmadebeforethephagetransfection.ThisdemonstratedtheexistenceofamessengermoleculemRNAwhichtransientlyassociateswithribosomesdirectingtheproductionofaspecificprotein.Thus,theproteinmadebyaribosomecouldchangedependingonwhichmRNAassociatedwiththeribosome.

Thethreestepsintranscriptionareinititation,elongation,andtermination.

InitiationinvolvesbindingoftheRNApolymerasetothepromotercausingalocalizedmeltingofaregionofabout12basepairs.ThepolymerasetheninitiatessynthesisofamessengerRNAchaincomplementarytothetemplatestrand.

TheelongationphasebeginsafterthefirstfewnucleotideshavebeenassembledandthepolymeraseleavesthepromotertomovedownstreamandcatalyzeelongationofthemRNAmolecule.

ParticularDNAsequencesatthe3’endofgenesareresponsiblefortermination,thefinalstageingenetranscription.RNApolymeraseinteractswiththesesequencesandtheresultislooseningoftheassociationbetweentheRNApolymerase,theRNA,anditsDNAtemplate.

A5S,a16Sanda23SribosomalRNAmoleculeareeachpresentintheE.coliribosome.The5Sand23SRNAsarepresentinthelarger50Ssubunitofthe70Sribosome.The16SribosomalRNAispresentinthesmaller30SsubunitoftheE.coliribosome.

ThecloverleafstructureofRNA

Theaminoacidattacheshere

AtRNAmoleculehastwofunctionalends,theaminoacidattachmentsite,andtheanticodonloop.ThekeytoitsabilitytoserveasanadaptorbetweenthecodonsofthemRNAandtheaminoacidsintheproteinisthespecificattachmentofaminoacidstotRNAs.ThecodonfortheaminoacidattachedtoatRNAiscomplementraytotheanticodononthatspecifictRNAmolecule.Thisspecificityofattachmentisaresultoftheactionofspecificaminoacyl-tRNAsynthetasescatalyzingthegenerationofchargedtRNAmolecules.Forexample,theenzymephenylalanine-tRNAsynthetase,attachestheaminoacidphenylalaninetotRNAmoleculesbearingtheanticodonGAAwhichwillbindtotheUUCorUUUphenylalaninecodonsensuringthecorrectadditionofthisaminoacidtothegrowingpeptidechain.Thus,tRNAmoleculesallowthegenerationofapolypeptidechaininwhichthelinearsequenceofaminoacidsisdeterminedbythesequenceofcodonsinthemRNA.

AsinglebasepairsubsititioninagenecanresultintheprematureterminationoftranslationofthemRNAfromthatgeneifthatsubstitutionresultsinthegenerationofthestopcodoninplaceofanaminoacidcodon.Forexample,thecodonUUGspecifiestheaminoacidleucine.AsinglebasepairsubstitutionresultinginalterationofthatcodontothestopcodonUAG,willleadtoprematureterminationofthepolypeptidechainattheleucineposition.

Thegeneticcodeisintheformof3nucleotidecodons,thesequentialorderofwhichdictatesthelinearsequenceofaminoacidsinapolypeptidechain.IfasinglebasepairinaDNAsequenceisdeleted,themutationwillaffect,notonlythatparticularcodon,butallsubsequentcodonssincethesewillnowhavelosttheiroriginaltripletgroupings.Thisiscalledaframeshiftmutationandfollowingtheframeshiftdifferentaminoacidswillbespecified.ThiscouldbychanceleadtotheprematureintroductionofastopcodoninthenucelotidesequencetherebyprematurelyhaltingtranslationofthecorrespondingmRNA.Considerthisexample:

Thesequence,ACUAUAGAAGGU,directsthesynthesisofthepeptidesequence:threonine,

isoleucine,glutamate,glycine.Deletingthe“A“atpositionfourwillresultinthesequence:ACUUAGAAGGU.Thesecondcodonisnowthestopcodon,UAG,andtranslationwillbeterminatedatthatpoint.

Asubstitutionofonenucleotideforanotherinthecodingregionofagenewillalterthethree-nucleotidesequenceinacodon.Giventhedegeneracyofthegeneticcode,thismaynotaltertheaminoacidspecifiedbythatcodon,butoftenitwill.Forexample,thecodonGGCspecifiestheaminoacidglycine.SubstutionofthesecondGwithaCwillresultinGCC,whichspecifiestheaminoacidalanine.

AnalyticalQuestions

Giventhisportionofabacterialgenewiththetemplatestrandonthebottom:

5’GTATCGTATGCATGCATCGTGAC3’

3’CATAGCATACGTACGTAGCACTG5’

ThemRNAtranscribedfromthisgenestartingfromthefirstTis:

5’AUCGUAUGCAUGCAUCGUGAC3’

Theinitiationcodon(AUG)isunderlined.

ChangingthefirstGi