基因的分子生物學(xué)

基因的分子生物學(xué)第1頁(yè)/共33頁(yè)2007-04-242Figure1.StructureofbacteriophageT4(modifiedfromEiserling&Black).Theproteinscomprisingthevirionarelabeledwiththeircorrespondinggenenumberorname.P.

G.

Leiman,S.

Kanamaru,V.

V.

Mesyanzhinov,F.

ArisakaandM.

G.

Rossmann.

StructureandmorphogenesisofbacteriophageT4.CellularandMolecularLifeSciences2003;

2356-2370

第2頁(yè)/共33頁(yè)2007-04-243Figure5.MorphogenesisofthebacteriophageT4virion.Theoverallassemblypathwaycanbedividedintothreeindependentstages:head,tail,andlongtailfiberassembly.Thechaperoninesandcatalyticproteinsareindicatedinbracketsneartheprotein,orassemblystep,thatrequiresthechaperonine.Knownproteinstoichiometriesaregivenassubscripts.Crystalstructuresofstructuralproteinsareshownasribbondrawings.第3頁(yè)/共33頁(yè)2007-04-244Figure8.Structureofthegp5-gp27complex.(a)Ribbonstereodiagram.Thethreegp5monomersarecoloredred,green,andblue.Thethreegp27monomersarecoloredyellow,gray,andpurple.TheK+ionwithingp5Cisshowninpink.The(PO4)–ishiddenbehindthelysozymedomain.(b)Thestructureofthegp27monomerwithitsfourdomainscoloredcyan,pink,lightgreen,andgoldalongthepolypeptidechain.(c)Topviewofthegp27cylindershowsthatthecyanandgreendomainsformahexagonaltorus.第4頁(yè)/共33頁(yè)2007-04-245Figure11.Infectionprocess.(a)T4phagerecognizestheE.coliLPSmoleculesusingtheLTFs.(b)Thephageattachesthebaseplatetothecellsurfaceinitiatingcontractionofthetailsheath.(c)Tailcontractioncausesthegp5needletopuncturetheoutercellmembrane.(d)Gp5Cdissociatesfromthetailtube,thusactivatingthethreelysozymedomains.(e)Thelysozymedomainscreateanopeninginthepeptidoglycanlayer.(f)Gp27associateswithareceptorontheinnermembrane,whichinitiatesreleaseofDNAintothecytoplasm.第5頁(yè)/共33頁(yè)2007-04-246Lyticinfection

ofabacteriumbyaphageendsinthedestructionofthebacteriumwithreleaseofprogenyphage.Lysis(裂解)

describesthedeathofbacteriaattheendofaphageinfectivecyclewhentheyburstopentoreleasetheprogenyofaninfectingphage(becausephageenzymesdisruptthebacterium'scytoplasmicmembraneorcellwall).Thesametermalsoappliestoeukaryoticcells;forexample,wheninfectedcellsareattackedbytheimmunesystem.Prophage

isaphagegenomecovalentlyintegratedasalinearpartofthebacterialchromosome.Lysogeny(溶源)

describestheabilityofaphagetosurviveinabacteriumasastableprophagecomponentofthebacterialgenome.Integration

ofviraloranotherDNAsequencedescribesitsinsertionintoahostgenomeasaregioncovalentlylinkedoneithersidetothehostsequences.Inductionofprophagedescribesitsentryintothelytic(infective)cycleasaresultofdestructionofthelysogenicrepressor,whichleadstoexcisionoffreephageDNAfromthebacterialchromosome.Theexcision

ofphageorepisomeorothersequencedescribesitsreleasefromthehostchromosomeasanautonomousDNAmolecule.Aplasmid

isacircular,extrachromosomalDNA.Itisautonomousandcanreplicateitself.Anextrachromosomalgenome

inabacteriumisaself-replicatingsetofgenesthatisnotpartofthebacterialchromosome.Inmanycases,thegenesarenecessaryforbacterialgrowthundercertainenvironmentalconditions.Anepisome

isaplasmidabletointegrateintobacterialDNA.Immunity

inphagesreferstotheabilityofaprophagetopreventanotherphageofthesametypefrominfectingacell.Itresultsfromthesynthesisofphagerepressorbytheprophagegenome.Immunity

inplasmidsdescribestheabilityofaplasmidtopreventanotherofthesametypefrombecomingestablishedinacell.Itresultsusuallyfrominterferencewiththeabilitytoreplicate.3.12.1Introduction第6頁(yè)/共33頁(yè)2007-04-247Figure12.1

Lyticdevelopmentinvolvesthereproductionofphageparticleswithdestructionofthehostbacterium,butlysogenicexistenceallowsthephagegenometobecarriedaspartofthebacterialgeneticinformation.第7頁(yè)/共33頁(yè)2007-04-248Figure12.2

Severaltypesofindependentgeneticunitsexistinbacteria.第8頁(yè)/共33頁(yè)2007-04-2493.12.2LyticdevelopmentisdividedintotwoperiodsEarlyinfection

isthepartofthephagelyticcyclebetweenentryandreplicationofthephageDNA.Duringthistime,thephagesynthesizestheenzymesneededtoreplicateitsDNA.Lateinfection

isthepartofthephagelyticcyclefromDNAreplicationtolysisofthecell.Duringthistime,theDNAisreplicatedandstructuralcomponentsofthephageparticlearesynthesized.第9頁(yè)/共33頁(yè)2007-04-2410Acascade

isasequenceofevents,eachofwhichisstimulatedbythepreviousone.Intranscriptionalregulation,asseeninsporulationandphagelyticdevelopment,itmeansthatregulationisdividedintostages,andateachstage,oneofthegenesthatareexpressedcodesforaregulatorneededtoexpressthegenesofthenextstage.Earlygenes

aretranscribedbeforethereplicationofphageDNA.Theycodeforregulatorsandotherproteinsneededforlaterstagesofinfection.Immediateearly

phagegenesinphagelambdaareequivalenttotheearlyclassofotherphages.TheyaretranscribedimmediatelyuponinfectionbythehostRNApolymerase.Delayedearly

genesinphagelambdaareequivalenttothemiddlegenesofotherphages.Theycannotbetranscribeduntilregulatorprotein(s)codedbytheimmediateearlygeneshavebeensynthesized.Middlegenes

arephagegenesthatareregulatedbytheproteinscodedbyearlygenes.SomeproteinscodedbymiddlegenescatalyzereplicationofthephageDNA;othersregulatetheexpressionofalatersetofgenes.Lategenes

aretranscribedwhenphageDNAisbeingreplicated.Theycodeforcomponentsofthephageparticle.3.12.3Lyticdevelopmentiscontrolledbyacascade第10頁(yè)/共33頁(yè)2007-04-2411Figure12.4

Phagelyticdevelopmentproceedsbyaregulatorycascade,inwhichageneproductateachstageisneededforexpressionofthegenesatthenextstage.第11頁(yè)/共33頁(yè)2007-04-2412Figure12.5

Controlatinitiationutilizesindependenttranscriptionunits,eachwithitsownpromoterandterminator,whichproduceindependentmRNAs.Thetranscriptionunitsneednotbelocatednearoneanother.Figure12.6

Controlatterminationrequiresadjacentunits,sothattranscriptioncanreadfromthefirstgeneintothenextgene.3.12.4Twotypesofregulatoryeventcontrolthelyticcascade第12頁(yè)/共33頁(yè)2007-04-2413Figure12.7

PhageT7containsthreeclassesofgenesthatareexpressedsequentially.Thegenomeis~38kb.Figure12.8

ThemapofT4iscircular.ThereisextensiveclusteringofgenescodingforcomponentsofthephageandprocessessuchasDNAreplication,butthereisalsodispersionofgenescodingforavarietyofenzymaticandotherfunctions.Essentialgenesareindicatedbynumbers.Nonessentialgenesareidentifiedbyletters.OnlysomerepresentativeT4genesareshownonthemap.3.12.5TheT7andT4genomesshowfunctionalclustering第13頁(yè)/共33頁(yè)2007-04-2414Figure12.9

ThephageT4lyticcascadefallsintotwoparts:earlyfunctionsareconcernedwithDNAsynthesisandgeneexpression;latefunctionsareconcernedwithparticleassembly.第14頁(yè)/共33頁(yè)2007-04-24153.12.6LambdaimmediateearlyanddelayedearlygenesareneededforbothlysogenyandthelyticcycleFigure12.10

Thelambdalyticcascadeisinterlockedwiththecircuitryforlysogeny.·Lambdahastwoimmediateearlygenes,Nandcro,whicharetranscribedbyhostRNApolymerase.·Nisrequiredtoexpressthedelayedearlygenes.·Threeofthedelayedearlygenesareregulators.·LysogenyrequiresthedelayedearlygenescII-cIII.·ThelyticcyclerequirestheimmediateearlygenecroandthedelayedearlygeneQ.第15頁(yè)/共33頁(yè)2007-04-2416·pNisanantiterminationfactorthatallowsRNApolymerasetocontinuetranscriptionpasttheendsofthetwoimmediateearlygenes.·pQistheproductofadelayedearlygeneandisanantiterminatorthatallowsRNApolymerasetotranscribethelategenes.Figure12.11

Thelambdamapshowsclusteringofrelatedfunctions.Thegenomeis48,514bp.3.12.7Thelyticcycledependsonantitermination第16頁(yè)/共33頁(yè)2007-04-2417Figure12.12

Phagelambdahastwoearlytranscriptionunits;inthe"leftward"unit,the"upper"strandistranscribedtowardtheleft;inthe"rightward"unit,the"lower"strandistranscribedtowardtheright.GenesNandcroaretheimmediateearlyfunctions,andareseparatedfromthedelayedearlygenesbytheterminators.SynthesisofNproteinallowsRNApolymerasetopasstheterminatorstL1totheleftandtR1totheright.第17頁(yè)/共33頁(yè)2007-04-2418Figure12.14

Thelambdaregulatoryregioncontainsaclusteroftrans-actingfunctionsandcis-actingelements.3.12.8Lysogenyismaintainedbyrepressorprotein第18頁(yè)/共33頁(yè)2007-04-24193.12.9RepressormaintainsanautogenouscircuitFigure12.16

Lysogenyismaintainedbyanautogenouscircuit(upper).Ifthiscircuitisinterrupted,thelyticcyclestarts(lower).第19頁(yè)/共33頁(yè)2007-04-2420·Arepressormonomerhastwodistinctdomains.·TheN-terminaldomaincontainstheDNA-bindingsite.·TheC-terminaldomaindimerizes.·BindingtotheoperatorrequiresthedimericformsothattwoDNA-bindingdomainscancontacttheoperatorsimultaneously.·Cleavageoftherepressorbetweenthetwodomainsreducestheaffinityfortheoperatorandinducesalyticcycle.Figure12.17

TheN-terminalandC-terminalregionsofrepressorformseparatedomains.TheC-terminaldomainsassociatetoformdimers;theN-terminaldomainsbindDNA.Immunity

inphagesreferstotheabilityofaprophagetopreventanotherphageofthesametypefrominfectingacell.Itresultsfromthesynthesisofphagerepressorbytheprophagegenome.Theimmunityregion

isasegmentofthephagegenomethatenablesaprophagetoinhibitadditionalphageofthesametypefrominfectingthebacterium.Thisregionhasagenethatencodesfortherepressor,aswellasthesitestowhichtherepressorbinds.3.12.10Therepressoranditsoperatorsdefinetheimmunityregion3.12.11TheDNA-bindingformofrepressorisadimer第20頁(yè)/共33頁(yè)2007-04-2421Figure12.18

Repressordimersbindtotheoperator.TheaffinityoftheN-terminaldomainsforDNAiscontrolledbythedimerizationoftheC-terminaldomains.Thebalancebetweenlysogenyandthelyticcycledependsontheconcentrationofrepressor.第21頁(yè)/共33頁(yè)2007-04-2422Figure12.19

Theoperatorisa17bpsequencewithanaxisofsymmetrythroughthecentralbasepair.Eachhalfsiteismarkedingreen.Basepairsthatareidenticalineachoperatorhalfareinred.3.12.12Repressorusesahelix-turn-helixmotiftobindDNA第22頁(yè)/共33頁(yè)2007-04-2423Figure12.20

Lambdarepressor''sN-terminaldomaincontainsfivestretchesofα-helix;helices2and3areinvolvedinbindingDNA.Figure12.21

Inthetwo-helixmodelforDNAbinding,helix-3ofeachmonomerliesinthewidegrooveonthesamefaceofDNA,andhelix-2liesacrossthegroove.第23頁(yè)/共33頁(yè)2007-04-2424Figure12.22

Twoproteinsthatusethetwo-helixarrangementtocontactDNArecognizelambdaoperatorswithaffinitiesdeterminedbytheaminoacidsequenceofhelix-3.Cro:antirepressorFigure12.23

AviewfromthebackshowsthatthebulkoftherepressorcontactsonefaceofDNA,butitsN-terminalarmsreacharoundtotheotherface.3.12.13TherecognitionhelixdeterminesspecificityforDNA第24頁(yè)/共33頁(yè)2007-04-2425·Repressorbindingtooneoperatorincreasestheaffinityforbindingasecondrepressordimertotheadjacentoperator.·Theaffinityis10×greaterforOL1andOR1thanotheroperators,sotheyareboundfirst.·CooperativityallowsrepressortobindtheO1/O2sitesatlowerconcentrations.Figure12.24

Eachoperatorcontainsthreerepressor-bindingsites,andoverlapswiththepromoteratwhichRNApolymerasebinds.TheorientationofOLhasbeenreversedfromusualtofacilitatecomparisonwithOR.3.12.14RepressordimersbindcooperativelytotheoperatorFigure12.25

Whentwolambdarepressordimersbindcooperatively,eachofthesubunitsofonedimercontactsasubunitintheotherdimer.第25頁(yè)/共33頁(yè)2007-04-2426·TheDNA-bindingregionofrepressoratOR2contactsRNApolymeraseandstabilizesitsbindingtoPRM.·Thisisthebasisfortheautogenouscontrolofrepressormaintenance.Figure12.26

Positivecontrolmutationsidentifyasmallregionathelix-2thatinteractsdirectlywithRNApolymerase.3.12.15RepressoratOR2interactswithRNApolymeraseatPRM第26頁(yè)/共33頁(yè)2007-04-2427·ThedelayedearlygeneproductscIIandcIIIarenecessaryforRNApolymerasetoinitiatetranscriptionatthepromoterPRE.·cIIactsdirectatthepromoterandcIIIprotectscIIfromdegradation.·TranscriptionfromPREleadstosynthesisofrepressorandalsoblocksthetranscriptionofcro.Figure12.27

RepressorsynthesisisestablishedbytheactionofcIIandRNApolymeraseatPREtoinitiatetranscriptionthatextendsfromtheantisensestrandofcrothroughthecIgene.3.12.16ThecIIandcIIIgenesareneededtoestablishlysogeny第27頁(yè)/共33頁(yè)2007-04-2428·PREhasatypicalsequencesat–10and–35.·RNApolymerasebindsthepromoteronlyinthepresenceofcII.·cIIbindstosequencesclosetothe–35region.ThePREpromoterhasapoorfitwiththeconsensusat–10andlacksaconsensussequenceat–35.ThisdeficiencyexplainsitsdependenceoncII.ThepromotercannotbetranscribedbyRNApolymerasealoneinvitro,butcanbetranscribedwhencIIisadded.Figure12.28

RNApolymerasebindstoPREonlyinthepresenceofcII,whichcontactstheregionaround-35.Figure12.29

PositiveregulationcaninfluenceRNApolymeraseateitherstageofinitiatingtranscription.3.12.17ApoorpromoterrequirescIIprotein第28頁(yè)/共33頁(yè)2007-04-2429·cII/cIIIcauserepressorsynthesistobeestablishedandalsotriggerinhibitionoflategenetranscription.·Establishmentofrepressorturnsoffimmediateanddelayedearlygeneexpression.·Repressorturnsonthemaintenancecircuitforitsownsynthesis.·LambdaDNAisintegratedintothebacterialgenomeatthefinalstageinestablishinglysogeny.Figure12.30

Acascadeisneededtoestablishlysogeny,butthenthiscircuitisswitchedoffandreplacedbytheautogenousrepressor-maintenanc