基因的分子生物學

基因的分子生物學第1頁/共48頁2007-04-112Thecodingstrand(Sensestrand)ofDNAhasthesamesequenceasthemRNAandisrelatedbythegeneticcodetotheproteinsequencethatitrepresents.Theantisensestrand(Templatestrand)ofDNAiscomplementarytothesensestrand,andistheonethatactsasthetemplateforsynthesisofmRNA.RNApolymerases

areenzymesthatsynthesizeRNAusingaDNAtemplate(formallydescribedasDNA-dependentRNApolymerases).Apromoter

isaregionofDNAwhereRNApolymerasebindstoinitiatetranscription.Startpoint(startsite)referstothepositiononDNAcorrespondingtothefirstbaseincorporatedintoRNA.Aterminator

isasequenceofDNAthatcausesRNApolymerasetoterminatetranscription.Atranscriptionunit

isthedistancebetweensitesofinitiationandterminationbyRNApolymerase;mayincludemorethanonegene.Upstream

identifiessequencesproceedingintheoppositedirectionfromexpression;forexample,thebacterialpromoterisupstreamofthetranscriptionunit,theinitiationcodonisupstreamofthecodingregion.Downstream

identifiessequencesproceedingfartherinthedirectionofexpression;forexample,thecodingregionisdownstreamoftheinitiationcodon.Aprimarytranscript

istheoriginalunmodifiedRNAproductcorrespondingtoatranscriptionunit.3.9.1Introduction第2頁/共48頁2007-04-113Figure9.1

ThefunctionofRNApolymeraseistocopyonestrandofduplexDNAintoRNA.Figure9.2

AtranscriptionunitisasequenceofDNAtranscribedintoasingleRNA,startingatthepromoterandendingattheterminator.Sequencespriortothestartpointaredescribedasupstream

ofit;thoseafterthestartpoint(withinthetranscribedsequence)aredownstream

ofit.第3頁/共48頁2007-04-114Withinthiscontext,therearetwobasicquestionsingeneexpression:·HowdoesRNApolymerasefindpromotersonDNA?Thisisaparticularexampleofamoregeneralquestion:howdoproteinsdistinguishtheirspecificbindingsitesinDNAfromothersequences?·HowdoregulatoryproteinsinteractwithRNApolymerase(andwithoneanother)toactivateortorepressspecificstepsintheinitiation,elongation,orterminationoftranscription?第4頁/共48頁2007-04-1153.9.2Transcriptionoccursbybasepairingina"bubble"ofunpairedDNAFigure9.3

DNAstrandsseparatetoformatranscriptionbubble.RNAissynthesizedbycomplementarybasepairingwithoneoftheDNAstrands.Figure9.4

Transcriptiontakesplaceinabubble,inwhichRNAissynthesizedbybasepairingwithonestrandofDNAinthetransientlyunwoundregion.Asthebubbleprogresses,theDNAduplexreformsbehindit,displacingtheRNAintheformofasinglepolynucleotidechain.第5頁/共48頁2007-04-116ThereisamajorchangeinthetopologyofDNAextendingover~1turn,butitisnotclearhowmuchofthisregionisactuallybasepairedwithRNAatanygivenmoment.CertainlytheRNA-DNAhybridisshortandtransient.Astheenzymemoveson,theDNAduplexreforms,andtheRNAisdisplacedasafreepolynucleotidechain.Aboutthelast25ribonucleotidesaddedtoagrowingchainarecomplexedwithDNAand/orenzymeatanymoment.Figure9.5

Duringtranscription,thebubbleismaintainedwithinbacterialRNApolymerase,whichunwindsandrewindsDNA,maintainstheconditionsofthepartnerandtemplateDNAstrands,andsynthesizesRNA.第6頁/共48頁2007-04-117Templaterecognition

beginswiththebindingofRNApolymerasetothedouble-strandedDNAatapromotertoforma"closedcomplex".ThenthestrandsofDNAareseparatedtoformthe"opencomplex"thatmakesthetemplatestrandavailableforbasepairingwithribonucleotides.ThetranscriptionbubbleiscreatedbyalocalunwindingthatbeginsatthesiteboundbyRNApolymerase.Initiation

describesthestagesoftranscriptionuptosynthesisofthefirstbondinRNA.ThisincludesbindingofRNApolymerasetothepromoterandmeltingashortregionofDNAintosinglestrands.Elongation

isthestageinamacromolecularsynthesisreaction(replication,transcription,ortranslation)whenthenucleotideorpolypeptidechainisbeingextendedbytheadditionofindividualsubunits.Terminationisaseparatereactionthatendsamacromolecularsynthesisreaction(replication,transcription,ortranslation),bystoppingtheadditionofsubunits,and(typically)causingdisassemblyofthesyntheticapparatus.3.9.3Thetranscriptionreactionhasfourstages第7頁/共48頁2007-04-118Figure9.8

Theβ(cyan)andβ’

subunit(pink)ofRNApolymerasehaveachannelfortheDNAtemplate.SynthesisofanRNAtranscript(copper)hasjustbegun;theDNAtemplate(red)andcoding(yellow)strandsareseparatedinatranscriptionbubble.PhotographkindlyprovidedbySethDarst.bacterialRNApolymerase3.9.5Amodelforenzymemovementissuggestedbythecrystalstructure第8頁/共48頁2007-04-119Figure9.9

TensubunitsofRNApolymeraseareplacedinpositionfromthecrystalstructure.Thecolorsofthesubunitsarethesameasinthecrystalstructuresofthefollowingfigures.Figure9.10

ThesideviewofthecrystalstructureofRNApolymeraseIIfromyeastshowsthatDNAishelddownstreambyapairofjawsandisclampedinpositionintheactivesite,whichcontainsanMg2+ion.PhotographkindlyprovidedbyRogerKornbergTheyeastenzymeisalargestructurewith12subunits.TensubunitsoftheyeastRNApolymeraseIIhavebeenlocatedonthecrystalstructure.

第9頁/共48頁2007-04-1110TheNobelPrizeinChemistry2006"forhisstudiesofthemolecularbasisofeukaryotictranscription"RogerD.KornbergUSAStanfordUniversity

b.1947第10頁/共48頁2007-04-1111Figure9.11

TheendviewofthecrystalstructureofRNApolymeraseIIfromyeastshowsthatDNAissurroundedby~270oofprotein.PhotographkindlyprovidedbyRogerKornberg.Figure9.12

DNAisforcedtomakeaturnattheactivesitebyawallofprotein.Nucleotidesmayentertheactivesitethroughaporeintheprotein.第11頁/共48頁2007-04-1112Figure9.13

AnexpandedviewoftheactivesiteshowsthesharpturninthepathofDNA.Figure9.14

Movementofanucleicacidpolymeraserequiresbreakingandremakingbondstothenucleotidesthatoccupyfixedpositionsrelativetotheenzymestructure.Thenucleotidesinthesepositionschangeeachtimetheenzymemovesabasealongthetemplate.第12頁/共48頁2007-04-1113Figure9.15

TheRNApolymeraseelongationcyclestartswithastraightbridgeadjacenttothenucleotideentrysite.Afternucleotideaddition,theenzymemovesonebasepairandbridgebendsasitretainscontactwiththenewlyaddednucleotide.Whenthebridgeisreleased,thecyclecanstartagain.第13頁/共48頁2007-04-1114Theholoenzyme(completeenzyme)isthecomplexoffivesubunitsincludingcoreenzyme(α2ββ’σ)factorthatiscompetenttoinitiatebacterialtranscription.Figure9.16

EubacterialRNApolymeraseshavefourtypesofsubunit;α,βandβ’

haveratherconstantsizesindifferentbacterialspecies,butσvaries

morewidely.3.9.6BacterialRNApolymeraseconsistsofmultiplesubunits第14頁/共48頁2007-04-1115Figure9.17

BoththetemplateandcodingstrandsofDNAarecontactedbytheβandβ’

subunitslargelyintheregionofthetranscriptionbubbleanddownstream.TheRNAiscontactedmostlyinthetranscriptionbubble.(UsuallythereisnodownstreamRNA,andcontactswithRNAdownstreamoccuronlyinthespecialcasewhentheenzymebacktracks.)第15頁/共48頁2007-04-1116Thecoreenzyme

isthecomplexofRNApolymerasesubunitsthatundertakeselongation.Itdoesnotincludeadditionalsubunitsorfactorsthatmayneededforinitiationortermination.Sigmafactor

isthesubunitofbacterialRNApolymeraseneededforinitiation;itisthemajorinfluenceonselectionofpromoters.Aloosebindingsite

isanyrandomsequenceofDNAthatisboundbythecoreRNApolymerasewhenitisnotengagedintranscription.Figure9.18

CoreenzymebindsindiscriminatelytoanyDNA.Sigmafactorreducestheaffinityforsequence-independentbinding,andconfersspecificityforpromoters.Butsigmafactoralsoconferstheabilitytorecognizespecificbindingsites.Theholoenzymebindstopromotersverytightly,withanassociationconstantincreasedfromthatofcoreenzymeby(onaverage)1000timesandwithahalf-lifeofseveralhours.3.9.7RNApolymeraseconsistsofthecoreenzymeandsigmafactor第16頁/共48頁2007-04-1117Anopencomplex

describesthestageofinitiationoftranscriptionwhenRNApolymerasecausesthetwostrandsofDNAtoseparatetoformthe"transcriptionbubble".Tightbinding

ofRNApolymerasetoDNAdescribestheformationofanopencomplex(whenthestrandsofDNAhaveseparated).Theternarycomplex

ininitiationoftranscriptionconsistsofRNApolymeraseandDNAandadinucleotidethatrepresentsthefirsttwobasesintheRNAproduct.Abortiveinitiation

describesaprocessinwhichRNApolymerasestartstranscriptionbutterminatesbeforeithasleftthepromoter.Itthenreinitiates.Severalcyclesmayoccurbeforetheelongationstagebegins.Figure9.19

RNApolymerasepassesthroughseveralstepspriortoelongation.Aclosedbinarycomplexisconvertedtoanopenformandthenintoaternarycomplex.3.9.8Theassociationwithsigmafactorchangesatinitiation第17頁/共48頁2007-04-1118Figure9.20

RNApolymeraseinitiallycontactstheregionfrom-55to+20.Whensigmadissociates,thecoreenzymecontractsto-30;whentheenzymemovesafewbasepairs,itbecomesmorecompactlyorganizedintothegeneralelongationcomplex.第18頁/共48頁2007-04-1119·TherateatwhichRNApolymerasebindstopromotersistoofasttobeaccountedforbyrandomdiffusion.·RNApolymeraseprobablybindstorandomsitesonDNAandexchangesthemwithothersequencesveryrapidlyuntilapromoterisfound.Figure9.21

CoreenzymeandholoenzymearedistributedonDNA,andverylittleRNApolymeraseisfree.3.9.10HowdoesRNApolymerasefindpromotersequences?第19頁/共48頁2007-04-1120Figure9.22

TheforwardrateconstantforRNApolymerasebindingtopromotersisfasterthanrandomdiffusion.Figure9.23

RNApolymerasebindsveryrapidlytorandomDNAsequencesandcouldfindapromoterbydirectdisplacementoftheboundDNAsequence.Figure9.24

RNApolymerasedoesnotslidealongDNA.第20頁/共48頁2007-04-1121·AchangeinassociationbetweensigmaandholoenzymechangesbindingaffinityforDNAsothatcoreenzymecanmovealongDNA.RNApolymeraseencountersadilemmainreconcilingitsneedsforinitiationwiththoseforelongation.Initiationrequirestightbindingonlytoparticularsequences(promoters),whileelongationrequirescloseassociationwithallsequencesthattheenzymeencountersduringtranscription.Figure9.25

Sigmafactorandcoreenzymerecycleatdifferentpointsintranscription.3.9.11SigmafactorcontrolsbindingtoDNA第21頁/共48頁2007-04-1122Conserved

positionsaredefinedwhenmanyexamplesofaparticularnucleicacidorproteinarecomparedandthesameindividualbasesoraminoacidsarealwaysfoundatparticularlocations.Aconsensussequence

isanidealizedsequenceinwhicheachpositionrepresentsthebasemostoftenfoundwhenmanyactualsequencesarecompared.The-10sequence

istheconsensussequencecenteredabout10bpbeforethestartpointofabacterialgene.ItisinvolvedinmeltingDNAduringtheinitiationreaction.The-35sequence

istheconsensussequencecenteredabout35bpbeforethestartpointofabacterialgene.ItisinvolvedininitialrecognitionbyRNApolymerase.3.9.12Promoterrecognitiondependsonconsensussequences第22頁/共48頁2007-04-1123Therearefour(perhapsfive)conservedfeaturesinabacterialpromoter:Thestartpoint;the–10sequence;the–35sequence;theseparationbetweenthe–10and–35sequences;and(sometimes)theUPelement-10sequence.ItsconsensusisTATAAT,andcanbesummarizedintheformT80A95T45A60A50T96-35sequence.TheconsensusisTTGACA;inmoredetailedform,theconservationisT82T84G78A65C54A45Figure9.26

Atypicalpromoterhasthreecomponents,consistingofconsensussequencesat-35and-10,andthestartpoint.第23頁/共48頁2007-04-1124Adownmutation

inapromoterdecreasestherateoftranscription.Anupmutation

inapromoterincreasestherateoftranscription.Figure9.27

The–35sequenceisusedforinitialrecognition,andthe–10sequenceisusedforthemeltingreactionthatconvertsaclosedcomplextoanopencomplex.3.9.13Promoterefficienciescanbeincreasedordecreasedbymutation第24頁/共48頁2007-04-1125Footprinting

isatechniqueforidentifyingthesiteonDNAboundbysomeproteinbyvirtueoftheprotectionofbondsinthisregionagainstattackbynucleases.Figure9.28

FootprintingidentifiesDNA-bindingsitesforproteinsbytheirprotectionagainstnicking.Figure9.29

OnefaceofthepromotercontainsthecontactpointsforRNA.3.9.14RNApolymerasebindstoonefaceofDNA第25頁/共48頁2007-04-1126·Negativesupercoilingincreasestheefficiencyofsomepromotersbyassistingthemeltingreaction.·Transcriptiongeneratespositivesupercoilsaheadoftheenzymeandnegativesupercoilsbehindit,andthesemustberemovedbygyraseandtopoisomerase.Figure9.30

Transcriptionmaygeneratemoretightlywound(positivelysupercoiled)DNAaheadofRNApolymerase,whiletheDNAbehindbecomeslesstightlywound(negativelysupercoiled).Transcriptionthereforehasasignificanteffectonthe(local)structureofDNA.Asaresult,theenzymesgyrase(introducesnegativesupercoils)andtopoisomeraseI(removesnegativesupercoils)arerequiredtorectifythesituationinfrontofandbehindthepolymerase,respectively.3.9.15Supercoilingisanimportantfeatureoftranscription第26頁/共48頁2007-04-1127Heatshockgenes

areasetoflocithatareactivatedinresponsetoanincreaseintemperature(andotherabusestothecell).Theyoccurinallorganisms.Theyusuallyincludechaperonesthatactondenaturedproteins.Figure9.31

Thesigmafactorassociatedwithcoreenzymedeterminesthesetofpromoterswheretranscriptionisinitiated.Figure9.32

Inadditionto

σ70,E.colihasseveralsigmafactorsthatareinducedbyparticularenvironmentalconditions.(Anumberinthenameofafactorindicatesitsmass.)3.9.16Substitutionofsigmafactorsmaycontrolinitiation第27頁/共48頁2007-04-1128Figure9.33

RseAissynthesizedasaproteinintheinnermembrane.ItscytoplasmicdomainbindstheσEfactor.Figure9.34

E.colisigmafactorsrecognizepromoterswithdifferentconsensussequences.第28頁/共48頁2007-04-11293.9.17SigmafactorsdirectlycontactDNAFigure9.35

AmapoftheE.coliσ70factoridentifiesconservedregions.Regions2.1and2.2contactcorepolymerase,2.3isrequiredformelting,and2.4and4.2contactthe–10and–35promoterelements.TheN-terminalregionprevents2.4and4.2frombindingtoDNAintheabsenceofcoreenzyme.Figure9.36

Aminoacidsinthe2.4α-helixofσ70contactspecificbasesinthecodingstrandofthe–10promotersequence.第29頁/共48頁2007-04-1130Figure9.38

Sigmafactorhasanelongatedstructurethatextendsalongthesurfaceofthecoresubunitswhentheholoenzymeisformed.Figure9.39

DNAinitiallycontactssigmafactor(pink)andcoreenzyme(gray).Itmovesdeeperintothecoreenzymetomakecontactsatthe–10sequence.Whensigmaisreleased,thewidthofthepassagecontainingDNAincreases.Figure9.37

TheN-terminusofsigmablockstheDNA-bindingregionsfrombindingtoDNA.Whenanopencomplexforms,theN-terminusswings20?away,andthetwoDNA-bindingregionsseparateby15?.第30頁/共48頁2007-04-1131Earlygenes

aretranscribedbeforethereplicationofphageDNA.Theycodeforregulatorsandotherproteinsneededforlaterstagesofinfection.Middlegenes

arephagegenesthatareregulatedbytheproteinscodedbyearlygenes.SomeproteinscodedbymiddlegenescatalyzereplicationofthephageDNA;othersregulatetheexpressionofalatersetofgenes.Lategenes

aretranscribedwhenphageDNAisbeingreplicated.Theycodeforcomponentsofthephageparticle.Acascade

isasequenceofevents,eachofwhichisstimulatedbythepreviousone.Intranscriptionalregulation,asseeninsporulationandphagelyticdevelopment,itmeansthatregulationisdividedintostages,andateachstage,oneofthegenesthatareexpressedcodesforaregulatorneededtoexpressthegenesofthenextstage.Figure9.40

TranscriptionofphageSPO1genesiscontrolledbytwosuccessivesubstitutionsofthesigmafactorthatchangetheinitiationspecificity.duringinfectionofB.subtilisbyphageSPO13.9.18Sigmafactorsmaybeorganizedintocascades第31頁/共48頁2007-04-1132Sporulation

isthegenerationofasporebyabacterium(bymorphologicalconversion)orbyayeast(astheproductofmeiosis).Thevegetativephase

describestheperiodofnormalgrowthanddivisionofabacterium.Forabacteriumthatcansporulate,thiscontrastswiththesporulationphase,whensporesarebeingformed.Aphosphorelay

describesapathwayinwhichaphosphategroupispassedalongaseriesofproteins.Figure9.41

Sporulationinvolvesthedifferentiationofavegetativebacteriumintoamothercellthatislysedandasporethatisreleased.Sporulationtakes~8hours.3.9.19Sporulationiscontrolledbysigmafactors第32頁/共48頁2007-04-1133Figure9.42

SporulationinvolvessuccessivechangesinthesigmafactorsthatcontroltheinitiationspecificityofRNApolymerase.Thecascadesintheforespore(left)andthemothercell(right)arerelatedbysignalspassedacrosstheseptum(indicatedbyhorizontalarrows).第33頁/共48頁2007-04-1134Figure9.43

σFtriggerssynthesisofthenextsigmafactorintheforespore(σG)andturnsonSpoIIRwhichcausesSpoIIGAtocleavepro-σE.Figure9.44

Thecrisscrossregulationofsporulationcoordinatestimingofeventsinthemothercellandforespore.第34頁/共48頁2007-04-1135Aterminator

isasequenceofDNAthatcausesRNApolymerasetoterminatetranscription.Readthrough

attranscriptionortranslationoccurswhenRNApolymeraseortheribosome,respectively,ignoresaterminationsignalbecauseofamutationofthetemplateorthebehaviorofanaccessoryfactor.Figure9.45

TheDNAsequencesrequiredforterminationarelocatedpriortotheterminatorsequence.FormationofahairpinintheRNAmaybenecessary.3.9.20BacterialRNApolymeraseterminatesatdiscretesites第35頁/共48頁2007-04-1136Intrinsicterminators

areabletoterminatetranscriptionbybacterialRNApolymeraseintheabsenceofanyadditionalfactors.Rho-dependent

terminatorsaresequencesthatterminatetranscriptionbybacterialRNApolymeraseinthepresenceoftherhofactor.Rhofactor

isaproteininvolvedinassistingE.coliRNApolymerasetoterminatetranscriptionatcertainterminators(calledrho-dependentterminators).Figure9.46

Intrinsicterminatorsincludepalindromicregionsthatformhairpinsvaryinginlengthfrom7-20bp.Thestem-loopstructureincludesaG-C-richregionandisfollowedbyarunofUresidues.Pointmutationsthatpreventterminationoccurwithinthestemregionofthehairpin.3.9.21TherearetwotypesofterminatorsinE.coli第36頁/共48頁2007-04-1137Figure9.47

Arut

sitehasasequencerichinCandpoorinGprecedingtheactualsite(s)oftermination.Thesequencecorrespondstothe3’endoftheRNA.3.9.22Howdoesrhofactorwork?第37頁/共48頁2007-04-1138Figure9.48

RhohasanN-terminalRNA-bindingdomainandaC-terminalATPasedomain.AhexamerintheformofagappedringbindsRNAalongtheexterioroftheN-terminaldomains.The5’endoftheRNAisboundbyasecondarybindingsiteintheinteriorofthehexamer.Figure9.49

RhofactorbindstoRNAatarutsiteandtranslocatesalongRNA,usingitshelicaseactivity,drivenbyATPhydrolysis,untilitreachestheRNA-DNAhybridinRNApolymerase,whereitreleasestheRNAfromtheDNA.第38頁/共48頁2007-04-1139Figure9.50

Theactionofrhofactormaycreatealinkbetweentranscriptionandtranslationwhenarho-dependentterminatorliessoonafteranonsensemutation.第39頁/共48頁2007-04-1140Antitermination

isamechanismoftranscriptionalcontrolinwhichterminationispreventedataspecificterminatorsite,allowingRNApolymerasetoreadintothegenesbeyondit.Antiterminationproteins

allowRNApolymerasetotranscribethroughcertainterminatorsites.Figure9.51

AntiterminationcanbeusedtocontroltranscriptionbydeterminingwhetherRNApolymeraseterminatesorreadsthroughaparticularterminatorintothefollowingregion.3.9.23Antiterminationisaregulatoryevent第40頁/共48頁2007-04