Chapter11 Structure and function of ribozymes.ppt

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1、Chapter11StructureandFunctionofRibozymes,Outline,DefinitionandDiscoveryofRibozymesComparisonbetweenProtein-basedandRNA-basedEnzymesTypesofribozymesSmallribozymesLargeribozymesMechanismofRibozymeCatalysisMechanismofsmallribozymecatalysisMechanismoflargeribozymecatalysisSignificanceofthediscoveryofrib

2、ozymesanditsapplications(self-study),DiscoveredbyTomCechandSydneyAltman(theNobelprizelaureates)RibozymesjustarecatalyticRNAmolecules,whichcancatalyzeanumberofthechemicalreactionsRibozymescanbenaturally-occurringorartificially-madeTwonaturally-occurringribozymesarepresentinalmosteveryorganism:RNasePa

3、ndRibosome,CechAltman,DiscoveryandDefinitionofRibozymes,ribozymerateenhancementscanbesubstantial.Forexample,therateconstantforchemistryoftheself-cleavingHDVribozymeisestimatedat102104s1,whichisclosetothemaximalcleavagerateofRNaseA.ribozymescanusecofactorsduringcatalysis,andtheycanbeswitchedonandoffb

4、ythebindingofsmall-moleculeallostericeffectors.ribozymes,likeproteincatalysts,foldintospecific3Dshapesthatcanharbordeepgroovesandsolvent-inaccessibleactivesites.Thesetertiarystructuresfacilitatecatalysisinpartbyorientingsubstratesadjacenttocatalyticgroupsandmetalions.Tofacilitatechemicaltransformati

5、ons,catalystsstabilizethetransitionstate.BothproteinandRNAcatalystsmayachievethisbyaddingorremovingprotonsduringareaction,orientingsubstratessothattheyareoptimallypositionedtoreact,andusingbindinginteractionsawayfromthereactionsitetoforceanunfavorablecontactthatisrelievedinthetransitionstate.,Common

6、pointsbetweenProtein-basedandRNA-basedEnzymes,ProteinsandRNAareverydifferentpolymers.Thephosphodiesterbackboneofnucleicacidsischarged,andthusRNAhasalargeelectrostaticcomponentinwhichmetalionshaveabigroletoplay.Therangeofchemicalreactionsthatarecatalyzedbyribozymesisratherlimited,andtherateenhancemen

7、tsarerelativelymodestbecausetherangeoffunctionalgroupsthatcanbecalledupontoparticipateinthechemistryislimitedcomparedtothesetofaminoacidside-chains.Thegreatmajorityofreactionscarriedoutbytheribozymesaretransesterificationreactions,althoughotherreactionscanbecatalyzedinafewcases.,DifferencesbetweenPr

8、otein-basedandRNA-basedEnzymes,DisadvantagesofRibozymecatalysis,RNAhasasmallrepertoireoffunctionalgroupsandtheseareembeddedinapoorlyconstrainedribosyl-phosphatebackboneheavywithnegativecharge.ThepKasofthebaseswouldappeartobeeithertoolow(3.5forAand4.2forC)ortoohigh(9.8forGand10.5forU)foruseinefficien

9、tgeneralacidorbasecatalysis.Asagroup,ribozymesarelessefficientcatalystsrelativetochemicallysuperchargedproteins,yetinmanycasesribozymesprovideenoughrateenhancementtohaveescapedreplacementbyproteinalternativesintheevolutionfromanRNAWorldtotheproteindominatedworldofmodernbiochemistry.,Naturallyoccurri

10、ngribozymesareclassifiedintotwodifferentgroups,thelargeandsmallribozymes,basedondifferencesinsizeandreactionmechanism.Artificialribozymesproducedbyinvitroselection.Thishasyieldedribozymesusingdifferentcatalyticmetalioncofactors.Inadditiontocleavagereactions,thecatalyzedreactionsincludephosphorylatio

11、n,ligation,polymerizationandtransesterification,andevenacarboncarbonbond.,TypesofRibozymes,SmallRibozymesandLargeRibozymes,Smallribozymesincludesthehammerheadandhairpinmotif,thehepatitisdeltavirus(HDV)RNA,theVarkudsatellite(VS)RNA,andtheglmSriboswitchribozyme.Theseribozymesrangeinsizefromabout40-154

12、nt.Thehammerhead,socalledforitsthreehelicesinaTshape,isthemostfrequentlyfoundcatalyticmotifinplantpathogenicRNAs,suchasviroids.Thehairpinribozymehasonlybeenfoundinsomevirusoids.TheHDVRNAisaviroid-likesatellitevirusoftheHBV.TheVSribozymeispartofalargerRNAthatistranscribedfromaplasmidfoundinthemitocho

13、ndriaofsomestrainsofNeurosporacrassa.TheglmSriboswitchribozymeisinvolvedinregulatingbacterialgeneexpression,Smallribozymes,Theyvaryinsizefromafewhundrednucleotidesuptoabout3000nt,andarefurtherdistinguishedfromthesmallribozymesbyallcleavingRNAtogenerate3-OHtermini,asopposedtoaproductwitha2,3-cyclicph

14、osphateandaproductwitha5-OHterminus.TheyincludeRNAcomponentofRNaseP,self-splicinggroupIandgroupIIintrons,theRNAcomponentsofthespliceosomeandtherRNAscatalyzingpeptidebondformationduringtranslation,Largeribozymes,ThecatalyticmechanismsemployedbyRNAarechemicallymorediversethaninitiallysuspected.Divalen

15、tmetalions,nucleobases,ribosylhydroxylgroups,andevenfunctionalgroupsonmetaboliccofactorsallcontributetothevariousstrategiesemployedbyRibozymes.ThelackofdiversefunctionalgroupsinRNAmoleculesandthepropensityforRNAtobindmetalionsledtoearlyhypothesesthatallribozymesmightactasmetalloenzymes,positioningme

16、talionsfordirectrolesincatalysis.ThisseemstobetrueforgroupIandgroupIIintronsandRNaseP.However,thoseribozymesthatperformsite-specificstrandscissionthehammerhead,hairpin,VSandHDVribozymesmayusediversecatalyticmechanisms,andnonehasemergedclearlyasametalloenzyme.,MechanismofRibozymeCatalysis,Mechanismof

17、smallribozymecatalysis,Smallribozymesperformsite-specificself-cleavage.Foundinviral,virusoid,orsatelliteRNAgenomes,theyprocesstheproductsofrollingcirclereplicationintogenome-lengthstrands.Thegeneralmechanismoftheseribozymesissimilartothatofmanyproteinribonucleasesinwhicha2oxygennucleophileattacksthe

18、adjacentphosphateintheRNAbackbone,resultingincleavageproductswith2,3-cyclicphosphateand5-OHUnlikeproteinribonucleases,however,ribozymescleaveonlyataspecificlocation,usingbase-pairingandtertiaryinteractionstohelpalignthecleavagesitewithinthecatalyticcore.Theevolutionarymaintenanceofthesesequencesmayb

19、eduetotheirsitespecificityandtothesimplicityandefficiencyofgenomeself-cleavage.,Internaltransesterificationreactioncatalyzedbythehammerhead,hairpin,HDV,VS,andglmSribozymes,Smallribozymesperformsite-specificself-cleavage,Mechanismoflargeribozymecatalysis,Incontrasttothesmallribozymes,thisclassofriboz

20、ymesrequiredivalentionsforthemtofunctionproficiently.CanbedifferentfromsmallribozymesbyallcleavingRNAtogenerate3-OHtermini.Agoodexample:GroupIintronsplicingpromotedbytwocatalyticmetalions,Largeribozymes,Smallribozymes,GroupIintronsplicingpromotedbytwocatalyticmetalions,TheRNAworldhypothesisproposest

21、hatself-replicatingRNAmoleculeswereprecursorstocurrentlife,whichisbasedonDNA,RNAandproteins.RNAstoresgeneticinformationlikeDNA,andcatalyzeschemicalreactionslikeanenzymeprotein.Itmay,therefore,haveplayedamajorstepintheevolutionofcellularlifeTheRNAworldwouldhaveeventuallybeenreplacedbytheDNA,RNAandpro

22、teinworldoftoday,likelythroughanintermediatestagesuchastheribosomeandribozymes,sinceproteinslargeenoughtoself-foldandhaveusefulactivitieswouldonlyhavecomeaboutafterRNAwasavailabletocatalyzepeptideligationoraminoacidpolymerization.DNAisthoughttohavetakenovertheroleofdatastorageduetoitsincreasedstabil

23、ity,whileproteins,throughagreatervarietyofaminoacids,replacedRNAsroleinspecializedbiocatalysis.TheRNAworldhypothesisissupportedbytheobservationthatmanyofthemostcriticalcomponentsofcells(thosethatevolvetheslowest)arecomposedmostlyorentirelyofRNA.ThiswouldmeanthattheRNAinmoderncellsisanevolutionaryremnantoftheRNAworldthatprecededours.Also,manycriticalcofactors(ATP,Acetyl-CoA,NADH,etc.)areeithernucleotidesorsubstancesclearlyrelatedtothem.,TheRNAworldhypothesis,TheRNAworldandthetransitiontothepresentDNA/RNA/proteinworld,