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Genetic Inquiry into Vaccinia Virus Intermediate and Late Gene Regulation

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Title:
Genetic Inquiry into Vaccinia Virus Intermediate and Late Gene Regulation
Creator:
CRESAWN, STEVEN GAINES ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
DNA ( jstor )
Enzymes ( jstor )
Genes ( jstor )
Genetic mutation ( jstor )
Genomes ( jstor )
In vitro fertilization ( jstor )
Peptide elongation factors ( jstor )
Polymerase chain reaction ( jstor )
RNA ( jstor )
Vaccinia virus ( jstor )

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Steven Gaines Cresawn. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
5/31/2006
Resource Identifier:
74655069 ( OCLC )

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Iowemanythankstothepeoplewhohaveencouragedandsupportedmethroughoutgraduateschoolandtheyearsprecedingit.Myparents,JamesandEileeneCresawn,deservemuchofthecredit.Theiremotionalandnancialsupporthavemademyeducationaljourneypossible,whiletheirloveandguidinghandsshapedthepersonthatIhavebecome.Theirtirelessworkethichasalwaysbeenaninspiration.Myolderbrother,RyanCresawn,hasbeenmyconstantfriend.Hisexamplehasalwaysbeenmyguide.Mywife,KerryCresawn,hasbeenmyconstantcompanionthroughoutourgraduateschooljourneys.Shehasgreatlysupportedandencouragedmethroughthemanypeaksandvalleysthatareapartofgraduateschoolandtaughtmethatthemostimportantmotivationcomesfromwithinoneself.Ourlifetogetherwithourdaughter,AbigailLeaCresawn,ismorethanIcouldhaveeverwanted.IamalreadyindebtedtoyoungAbbieformakingsurethatnothinginourlivesisevertakenmoreseriouslythanitshouldbe.Forthelast6years,Dr.RichCondithasbeenmymentorandhasbecomemyfriend.Ihavebenetedgreatlyfromhisselessdedicationandloyaltytohisstudents,andIhavelearnedmuchfromhisteachinginthelab,theclassroom,andbeyond.Richhastaughtmehowtodoresearch,howtocommunicateideasandresultseectively,andhowtobalancethedemandsofajobinsciencewiththemorecriticalworkofbeingagoodhusbandandfather.Forallowingmetheopportunitytoworkinhislab,Iwillalwaysbegrateful.Additionally,anumberofmembersoftheConditlabdeservespecialthanks.FormergraduatestudentsDr.CariLacknerandDr.DonLatnerprovidedexcellent iv

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v

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page ACKNOWLEDGMENTS ............................. iv LISTOFTABLES ................................. ix LISTOFFIGURES ................................ x ABSTRACT .................................... xii CHAPTER 1INTRODUCTION .............................. 1 1.1Poxviruses ............................... 1 1.2LifeCycle ............................... 2 1.2.1CellEntry ........................... 2 1.2.2EarlyTranscriptionintheCoreParticle ........... 5 1.2.3DNAReplication ....................... 6 1.2.4IntermediateandLateTranscription ............. 7 1.2.5Morphogenesis ......................... 7 1.2.6VirionTrackingandExit .................. 8 1.3Transcription ............................. 8 1.3.1Prokaryotes .......................... 10 1.3.2Eukaryotes ........................... 10 1.3.3VacciniaVirus ......................... 11 1.4Structure ................................ 19 1.5Elongation ............................... 22 1.5.1ElongationMechanism .................... 23 1.5.2RecoveryfromArrest ..................... 26 1.5.3RecoveryfromPausing .................... 27 1.6Termination .............................. 36 1.6.1IntrinsicTermination ..................... 37 1.6.2Rho-DependentTermination ................. 39 1.6.3MutationFrequencyDecline ................. 43 1.6.4RibonucleicAcidTracking1 ................ 44 1.6.5TranscriptionTerminationFactor2 ............. 45 1.6.6VacciniaVirusEarlyTermination .............. 46 1.6.7VacciniaVirusIntermediateandLateTermination ..... 48 vi

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....................... 58 2.1CellsandViruses ........................... 58 2.2PolymeraseChainReaction ..................... 58 2.3DNASequencing ........................... 59 2.4MarkerRescueMapping ....................... 59 2.5RNAIsolation ............................. 60 2.6NorthernBlotting ........................... 60 2.7HomologyModeling .......................... 61 2.8Error-PronePCR ........................... 62 3MAPPINGANDPHENOTYPICANALYSISOFSPONTANEOUSISATIN--THIOSEMICARBAZONE-RESISTANTVACCINIAVIRUSMUTANTS ............................ 63 3.1Introduction .............................. 63 3.2Results ................................. 64 3.2.1PlaqueAssay .......................... 64 3.2.2GeneticMapping ....................... 66 3.2.3InVivoAnalysisofTranscriptionElongation ........ 72 3.2.4InVitroAnalysisofTranscriptionElongation ........ 77 3.2.5HomologyModelingofRNAPolymeraseSubunits ..... 78 3.3Conclusions .............................. 82 4TARGETEDAPPROACHTOIDENTIFYTRANSCRIPTIONREGULATORS .............................. 88 4.1Introduction .............................. 88 4.2Results ................................. 89 4.2.1ScreeningCandidateGenes .................. 89 4.2.2SpecicityoftheTargetingApproach ............ 92 4.2.3IsolationofanIBT-ResistantH5RMutant ......... 92 4.2.4Error-PronePCRMutantGenotypes ............ 94 4.2.5DistinguishingbetweenIBT-ResistanceandIBT-Dependence 96 4.2.6HomologyModeling ...................... 96 4.2.7Conclusions .......................... 99 5DISCUSSION ................................. 101 5.1Introduction .............................. 101 5.2A18 ................................... 102 5.3G2andJ3 ............................... 103 5.4H5 ................................... 104 5.5RNAPolymerase ........................... 106 5.6ModelofIntermediateandLateTranscription ........... 107 5.7Conclusions .............................. 108 vii

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................................... 110 BIOGRAPHICALSKETCH ............................ 137 viii

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Table page 1{1Vacciniavirustranscriptionfactors .................... 18 3{1GenotypesofspontaneousIBT-resistantmutants ............ 76 4{1Genotypesoferror-pronePCRmutants ................. 96 ix

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Figure page 1{1Thepoxviruslifecycle .......................... 3 1{2Modelforvacciniavirusearlytranscriptiontermination ........ 47 1{3Isatin-thiosemicarbazone(IBT)andtwoderivativeswithanti-poxviralactivity ................................. 51 1{4Modelforvacciniavirusintermediateandlatetranscriptiontermination 57 3{1PlaqueassayofIBT-resistantmutantsinthepresenceandabsenceofIBT ................................... 65 3{2MarkerrescueofviruseswithmutationsinG2R 68 3{3PrimerpairsusedtoconstructPCRproductsformarkerrescue. ... 69 3{4InitialmarkerrescuemappingofDL1-3 ................. 70 3{5MarkerrescuemappingofDL1-3totheJ6Rgene ........... 71 3{6InitialmarkerrescuemappingofDL3-2 ................. 73 3{7MarkerrescuemappingofDL3-2totheJ6Rgene ........... 74 3{8MarkerrescuemappingofDL10-7.1totheJ6Rgene ......... 75 3{9NorthernanalysisofIBT-resistantmutantRNAwithaK2L-specicriboprobe ................................ 79 3{10NorthernanalysisofIBT-resistantmutantRNAwithanA10L-specicriboprobe ................................ 80 3{11NorthernanalysisofIBT-resistantmutantRNAwithanA18R-specicriboprobe ................................ 81 3{12Structuralmodelofthevacciniavirusrpo132proteinwithpositionoftheIBTr90mutationindicated ..................... 83 3{13Structuralmodelofthevacciniavirusrpo147proteinwithpositionsoftheDL1-3/DL10-7.1andDL3-2mutationsindicated ....... 84 4{1Ascreenofvacciniavirusgenesfortheirinvolvementinregulationofintermediateandlatetranscription .................. 91 x

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.............. 93 4{3Amutantconstructedbyerror-pronePCRmutagenesisoftheH5RgenemapstoH5R 95 4{4G2R-R1,A24R-R1,A24R-R2,andH5R-R1areIBT-resistant,whileG2R-D1isIBT-dependent ....................... 97 4{5Structuralmodelofthevacciniavirusrpo132proteinwithpositionsofA24R-R1andA24R-R2mutationsindicated ........... 98 5{1SummaryofallmappedG2Rmutants ................. 105 5{2Modelforvacciniavirusintermediateandlatetranscriptionelongationandtermination ............................. 109 xi

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xii

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xiii

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1.1 PoxvirusesPoxvirusesowemuchoftheirnotorietytothefactthatvariolavirus,amemberofthefamily,isthecausativeagentofsmallpox.Smallpoxisadevastatingdiseasethatravagedthehumanpopulationaroundtheglobeforcenturies,scarringandkillinguntoldnumbers.Inthe20thcenturyalone,smallpoxisthoughttohaveclaimedapproximately500millionlives[ 1 ].In1953aplanwasconceivedtoeradicatesmallpoxglobally.Attheheartofthisplanwasvaccination,thedeliberateinfectionofhealthyindividualswithanother,lesspathogenicpoxvirus.Cowpoxviruswaslikelytheoriginalvaccinestrain,butlatervacciniaviruswasused.Thisprocedure,originallydevelopedbyEdwardJennerin1796,washighlyeectiveatstemmingthespreadofthedisease.Vaccinationwaseectiveforseveralreasons,includingeaseofadministrationandthermostabilityofthevaccine.Ofparamountimportance,however,wastheimmunologicalcross-reactivityamongmembersofthepoxvirusfamily.Thesefactors,theplanitself,andthediligenceofthosewhocarrieditoutresultedinwhatmaybethesinglegreatesttriumphofmodernmedicalscience.OnDecember9,1979,theWorldHealthOrganizationcertiedthatsmallpoxhadbeeneradicated.Whileinterestinvacciniavirusbeganasaresultofitsuseinvaccinationagainstsmallpox,itwassustainedbytheuniquebiologicalpropertiesofthepoxvirusfamily.Membersofthepoxvirusfamilyenjoyadegreeofautonomythatisunmatchedbyanyothervirusfamily.Poxvirusesarelarge,double-strandedDNA(dsDNA)-containingvirusesthatreplicateexclusivelyinthecytoplasmofinfectedcells.Theycontainalargegenome,varyinginsizefrom134kbpinbovinepapular 1

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stomatitisvirus[ 2 ]to360kilobasepairs(kbp)incanarypox[ 3 ].Thearchetypalpoxvirus,vacciniavirus,containsa195kbpgenome.ThecytoplasmiclifestyleandDNAgenomerequirepoxvirusestoencodetheenzymesrequiredforgenomereplicationandtranscription,ataskthatisfacilitatedbythelargecodingcapacityoftheirgenomes.BecausepoxvirusesperformDNAandRNAsynthesisinthecytoplasm,freefromtheenormouscomplexityofthenucleus,theyhavelongbeenusedasmodelsforstudyingDNAreplicationandtranscription.Becauseofitsrichhistory,therelativesafetyinwhichitcanbestudied,anditseaseofgrowthincellculture,vacciniavirushasbeenandcontinuestobethedefactosubjectforstudiesofpoxviralgenetics,biochemistry,and(toalesserextent)hostinteractions.Theremainderofthissectionaddressesspecicaspectsofvacciniavirusbiology,thoughmanyofthecommentshereapplytootherpoxvirusesaswell.Abriefdiscussionofthevacciniaviruslifecycleispresentedrstandisfollowedbyamoredetailedreviewofthevacciniavirustranscriptionparadigm. 1.2 LifeCycleThevacciniaviruslifecycleconsistsofaseriesoftightlyregulated,coordinatedevents.Theseincludecellentry,uncoating,earlygenetranscription,DNAreplication,intermediategeneexpression,lategeneexpression,andvirionmorphogenesis.Eachoftheseprocessesiscomplex,andtheyareunderstoodtovaryingdegrees.Theareasinwhichthereistheleastconsensusofopinionarevirusentryandmorphogenesis.Morphogenesishasbeenthesubjectofcontinueddebatefordecades. 1.2.1 CellEntryEntryofvacciniavirusintothehostcellcanbeaccomplishedbythreedierentformsofthevirion.Theseincludeintracellularmaturevirus(IMV),cell-associatedenvelopedvirus(CEV),andextracellularenvelopedvirus(EEV).TheCEVandEEVformsareidentical,exceptthatCEVisattachedtothecellsurface,while

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Figure1{1:Thepoxviruslifecycle.Entryoftheintracellularmaturevirus(IMV)formofthevacciniavirionintothecelloccursbymembranefusion.(Forsimplicity,theentryofotherinfectiousvirionformsisomitted.)Afterapartialuncoatingstep,earlygenetranscriptioniscarriedoutwiththecontextofthepartiallyintactvirioncoreparticle(shownsurroundedbyadottedline).EarlygeneproductsplayanessentialroleinDNAreplicationandthesubsequenttranscriptionofintermediategenes.Intermediategeneproductsarerequiredforlategenetranscription.Lategeneproductsincludefactorsnecessaryforearlygenetranscriptioninthesubsequentlyinfectedcell.TheyareincorporatedalongwithviralDNAintoIMVvirionsastheygothroughanumberoftransformationsintheviroplasmfromcrescentstoimmaturevirions(IV)toimmaturevirionswithnucleoids(IVN)andnallytoIMV.TrackingofIMValongmicrotubulesisnecessaryforadoublemembranewrappingthroughtheGolgiorearlyendosomes(notshown)toformintracellularenvelopedvirus(IEV).AdditionalmovementalongmicrotubulesbringstheIEVtothecellsurface,whereanotherfusioneventleavescell-associatedenvelopedvirus(CEV)attachedtothecellsurface.ThisCEVcanpolymerizeintracellularactinlamentstopropelitselfintoanadjacentcellordetachfromthemembranetobecomeextracellularenvelopedvirus(EEV).

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EEVisnot.Afourthform,intracellularenvelopedvirus(IEV)isanintermediatebetweenIMVandCEV.Themechanismofcellentryvariesaccordingtoviriontype,butregardless,itisincompletelyunderstood.WhatisknownaboutentrybyIMVandCEV/EEVisdiscussednext,whiletheformationoftheseparticleswillbedescribedlater(Section1.2.5).AlthoughIMVisthemost-abundantvirionformandtheeasiesttoisolate,manyquestionsremainaboutitsmechanismofcellentry.CentraltothisdebateisuncertaintyaboutthenumberofmembranessurroundingtheIMVparticle.Clearly,thenumberofmembranesiscentraltothequestionofhowthevirustraversestheplasmamembrane,andthedisagreementonthisfundamentalpointhighlightshowmuchislefttolearnaboutvirusentry.Onthequestionofmembranenumber,therearetwogroups:thosewhobelievethatIMVissurroundedbyonemembrane,andthosewhobelievethattherearetwomembranes.IfIMVhasonemembrane,thenitlikelyentersthecellbyfusion[ 4 ],thoughothershavesuggestedphagocytosis,followedbyfusionoftheviralmembranewithmembraneofthephagosomevesicle[ 5 ].Recentelectronmicroscopy(EM)andconfocalmicroscopydataprovidestrongevidencethatthereisonemembrane,andthatIMVentersthehostcellviafusion(GeoreySmith,personalcommunication).ThisworkdoesnotanswerthequestionssurroundingtheoriginofsuchanIMVbilayer.PreviousstudiessuggestedadoublebilayersurroundingIMVthatmightbeformedfromlipidintheendoplasmicreticulumandGolgiintermediatecompartment(ERGIC)[ 6 ].Suchamodelraisesthequestionofhowtheextramembraneisshedduringvirusentry,however.AswiththeentryofIMV,theentryofEEVispoorlyunderstood.SpecicpropertiesofEEVmakeitmorediculttostudythanIMV.First,itisproducedinlowerquantitythanIMV.Second,preparationsofEEVareoftencontaminatedwithIMV,asdisruptionofthefragileEEVoutermembraneproducesanIMV

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particle.Regardless,whatdoesseemclearisthatIMVandEEVbinddierentcellularreceptors[ 7 ].Also,asasimplematteroftopology,EEVcannotusethesameentrymechanismasdoesIMV,sinceEEVcontainsanadditionalmembranethatmustbeshedbeforethecoreparticlecanbereleased.AplausiblemodelforEEVentryisthatofIchihashi[ 8 ].Inthismodel,EEVparticlesarepinocytosed.WhentheEEV-containingvesiclesareacidied,theouterEEVmembraneisruptured,allowingtheremainingIMVparticletofusewiththevesicularmembrane.Thisreleasesthecoreparticleintothecytoplasm. 1.2.2 EarlyTranscriptionintheCoreParticleAftervirusentry,transcriptionofearlymRNAoccurs.Thishappenswithinthecoreparticle,withthenewlysynthesizedmRNAsbeingextrudedthroughthecorewall.Earlytranscriptionreliesentirelyonenzymescarriedintothecellinthevirion.Theseincludethenine-subunitRNApolymerase,theearlygene-specictranscriptionfactor(VETF),themRNAcappingenzyme,andthepoly(A)polymerase.Alsopresentaretheearlygeneterminationfactors,vacciniaterminationfactor(VTF)andnucleosidetriphosphatephosphohydrolaseI(NPH-I).TheproductsofearlygenetranscriptionincludethefactorsrequiredforbothintermediategenetranscriptionandDNAreplication,andthusearlytranscriptionsetsthestageforbothoftheseprocesses.Asisdescribedbelow(Sections1.6.3and1.6.5),helicasesplayimportantrolesinregulatingtranscriptiontermination.However,avacciniavirusRNAhelicaseplaysaninterestingroleintranscriptionuniquelyinthecontextofthevacciniaviruscoreparticle.Thisenzyme,theDNA-orRNA-dependentATPasenucleosidetriphosphatephosphohydrolaseII(NPH-II),isimplicatedinextrusionofRNAthroughthecorewall.Consistentwiththismodel,NPH-IIhasRNA/RNAandDNA/RNAhelicaseactivity,butcannotunwindduplexDNA[ 9 , 10 ].Also,NPH-IIisessentialfortheprocessofearlytranscription,butspecicallywithinthecontext

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oftheviruscoreparticle.ThishasledtothesuggestionthatNPH-IIfacilitatesextrusionofmRNAfromtheviruscorebyusingitshelicaseactivity,whileitselfbeingessentiallyxedinlocationrelativetothecoreparticle[ 11 ].Activityofthistypemorecloselytsthedescriptionofatranslocasethanahelicase(seeSection1.6.2forcommentary),thoughNPH-IIclearlypossesseshelicaseactivityinvitro. 1.2.3 DNAReplicationThereplicationofvacciniavirusDNAtakesplaceinfocicalledviroplasmorviralfactorieswhichpresumablyformatthesiteofDNAunpackagingfromviralcores.Viroplasmisanamorphous,electron-denseregioninthecytoplasmandisdevoidofcellularorganelles.ItisthesiteforDNAreplication,intermediateandlatetranscription,andtheearlystagesofmorphogenesis[ 12 ].VacciniavirusDNAreplicationoccursthroughconcatamericintermediates.Replicationbeginsafterviraluncoating,allowingaccessoftheviralgenometothecytoplasm.Thisgenomeisasingle,lineardouble-strandedDNAmoleculewithcovalentlyclosedhairpintermini.Thus,inasenseitisasinglecontinuousstringofnucleicacid,afactthathasimportantimplicationsforDNAreplication.Onceuncoatingoccurs,anickiscreatednearoneorbothhairpins,allowingreplicationusingthe3'-OHgroupasaprimer.Bystranddisplacement,thisDNAreplicationresultsintheformationoftail-tailconcatemersthataresubsequentlycleavedtoformgenome-lengthDNA[ 13 ].ThevacciniavirusDNApolymeraseistheproductoftheE9Lgene[ 14 ].This110kDaenzymeisahomologofeukaryoticandDNApolymerasesandherpesviralDNApolymerases.E9hasbeenthesubjectofanexhaustiveseriesofbiochemicalandgeneticexperimentstocharacterizeitsactivity,functionaldomains,andbindingpartners.Thisworkhasbeenfacilitatedbythestudyofdrug-resistantmutants.

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ThereplicationofDNAdependsonthepolymeraseitself,andalsoonatleastfourotherfactors.Thesefactors(theD4uracilDNAglycosylase,B1kinase,andD5NTPase,andA20processivityfactor)havebeencharacterizedtovaryingdegrees[ 12 , 15 ].Mostofthosestudiesusedtemperature-sensitivevacciniavirusmutants. 1.2.4 IntermediateandLateTranscriptionAftertheinitiationofDNAreplication,intermediatetranscriptionensues[ 16 ].Latetranscriptionfollowsintermediatetranscription(Section1.3.3)[ 17 , 18 ].Briey,intermediatetranscriptionproducesproteinsrequiredforlatetranscription,andlatetranscriptionproducesfactorsrequiredformorphogenesisaswellasearlytranscriptionfactorsthatarepackagedintovirionsandusedinthesubsequentlyinfectedcell.Regulationofintermediateandlategenetranscriptionelongationwasthefocusofourstudyandisfundamentallydierentfromthatofearlytranscription. 1.2.5 MorphogenesisMorphogenesisistheprocessofvirionassembly.Severalstructuralintermediatesinthisprocessarediscernibleusingelectronmicroscopy:crescents,immaturevirions(IV),immaturevirionswithnucleoid(IVN),andIMV.Asthenameimplies,crescentshaveasemicircularshape,thoughoneshouldbearinmindthatwhiletheyappearcrescent-shapedundertheelectronmicroscope,thisisprobablynotanaccuraterepresentationoftheirthree-dimensionalstructure.Crescentsarecomposedofbothproteinandlipid,thoughtheoriginoftheirmembranouscomponentisunknown.Earlystudiessuggestedthatcrescentmembranesaresynthesizeddenovo,thoughlaterworkarguedthattheyarederivedfromcellularmembranes.Immaturevirionsareseenaftertheappearanceofcrescentsandarethoughttoformfromcrescentsgrowingandreshapingtoformenclosedspheres.ImmaturevirusparticlescontaintheDNAgenome,thoughtheexactmechanism

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forpackagingthatgenomeisunclear.Regardless,theDNAcondensestoformanelectron-densenucleoidwithintheIVparticle,thustransitioningtheparticlefromIVtoIVN.Finally,theIVNparticleundergoesamajorstructuralreorganizationtoproduceIMV.Thisstepisundoubtedlythebiggestmysteryinthemorphogenesisprocess. 1.2.6 VirionTrackingandExitAfterIMVformation,trackingoftheparticlealongmicrotubulesthroughtheGolgiapparatusorearlyendosomesresultsinthewrappingoftwoadditionalmembranestoformIEV,andonexitfromthecell,CEVandEEV.MovementtothesiteofIEVwrappingandmovementfromtheretothecellsurfacedependonmicrotubules,whileprotrusionoftheCEVparticleintoanadjacentcelldependsinsteadonpolymerizationofactinlamentsbelowthecellsurface[ 19 , 20 , 21 ]. 1.3 TranscriptionAnorganism'sgenomeencodestheinformationneededtosynthesizemacromoleculesthatcarryouttaskswithinthecell.Broadlyspeaking,therearetwosuchtypesofmacromolecules:RNAandprotein.ThethreemostwidelyrecognizedtypesofRNAareribosomalRNA(rRNA),transferRNA(tRNA),andmessengerRNA(mRNA).Eachoftheseplaysavitalroleinconvertingtheinformationcontainedinageneintotheproteinitencodes.ThevarioustypesofRNAmoleculesareassembledfrommanysubunits(calledribonucleotides)intolongpolymersbyanenzyme(calledRNApolymerase).ThisenzymeusesasatemplateforRNAsynthesistheorganism'sgenome,whichcanbemadeofeitherDNAorRNA.Thus,twofundamentallydistincttypesofRNApolymeraseexist:theDNA-dependentRNApolymeraseandtheRNA-dependentRNApolymerase.TheformerisproducedbyallcellularorganismsandisproducedorhijackedbytheDNAvirusesthatinfectthosecells,whilethelatterisconnedtoasubsetofviruseswhosegenomeiscomposedofRNA.OftheDNA-dependent

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RNApolymerases,therearetwoprimarytypes.Oneisasingle-subunitenzymefoundinbacteriophage,whiletheotherisalarge,well-conserved,multi-subunitenzymefoundinallmannerofcreatures,includingthepoxvirusfamily.Theremainderofthisdiscussionfocusesonthemulti-subunitDNA-dependentRNApolymerases.Thesynthesisofgeneproductsisregulatedatmanystages.Transcription,theprocessofRNAsynthesis,andtranslation,theprocessofsynthesizingproteinfrommRNA,areeachsubjecttomultipleregulatorymeans.ModicationofRNAduringoraftertranscription,degradationofRNA,aswellasmodicationofproteinsduringoraftertheirsynthesisareallwaystoregulatetheabundanceandfunctionofthesemolecules.However,theprocessoftranscriptionitselfrepresentsthekeyregulatorystepinproteinsynthesis.Transcriptioncanbesubdividedintoaseriesofsteps:promoterrecognition,initiation,elongation,andtermination[ 22 ].Apromotersignalsavalidlocationforthestartoftranscription,anditprovidesaneededassemblypointforRNApolymeraseandvarioustranscriptionfactorsrequiredtoinitiateRNAsynthesis.Oncebegun,therateandextentofRNAsynthesisismediatedbytwoopposinggroupsoffactors.Therstgroupcomprisespositivelyactingelongationfactors.ThesepromotecontinuedsynthesisofRNA.Conversely,thesecondgroupcomprisesnegativelyactingelongationfactors.Thesepromotecessationoftranscription,ortermination.Thesubtleinterplayofthesetwogroupsoffactors(coupledwiththeirinteractionswithvariouscis-actingregulatoryelementsburiedwithinthegenome)ultimatelydeterminesthelengthsofindividualRNAmolecules.Liketheirmorecomplexcellularcounterparts,virusesdependonnumerousregulatorymechanismstodirectthecorrecttranscriptionoftheirgenes.Theseregulatorymechanisms,liketheviralRNApolymerasesthemselves,areasexquisiteastheyarevaried.Somearenearlyuniversallyconservedthroughoutnature,others

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areborrowedfromtheirhosts,andmanyothersareuniquetotheindividualvirusfamiliesthemselves.Sections1.3.1,1.3.2,and1.3.3compareandcontrasttheenzymaticmachineryandregulatorymechanismsusedbyselectedprokaryotes,eukaryotes,andvacciniavirus.AlthoughtheorganismsselectedarepresentedforthediversityofstrategiestheyusetosynthesizeRNA,theysharemuchincommon.Thetranscriptioninitiationcomplexandmechanismofinitiationarethefocusofthissection.Aselongationandterminationaretheprimaryfocusofourstudy,theywillbediscussedingreaterdetail(Sections1.5and1.6). 1.3.1 ProkaryotesProkaryotesencodeasingleRNApolymerasethattranscribestheirentirerepertoireofgenes[ 23 ].Thisenzymeiscomposedofafour-subunit\core"andanadditionalsubunit,,thatprovidespromoterspecicity.Thecorecomprisesoneeachof'andandtwosubunits.Thesubunitmaybeoneofanumberofproteins,eachprovidingspecicityforadierentclassofpromoters. 1.3.2 EukaryotesEukaryotesencodethreeRNApolymerases(I,II,andIII).EachisresponsibleforthetranscriptionofaspecicclassofRNA.TheseenzymesarecloselyrelatedtooneanotherandmoredistantlyrelatedtoprokaryoticRNApolymerase.Theeukaryoticpolymerasesconsistoftwolargesubunitsandacollectionofsmallersubunits.Thelargestandsecond-largestsubunitsarehomologsofbacterial'and,respectively.Homologsofthebacterialsubunitalsoexistintheeukaryoticpolymerases.Additionally,sixsubunitsaresharedamongthethreeeukaryoticenzymes.Anumberofothersubunitsarenotshared[ 24 ]. RNApolymeraseII.Thetranscriptionofallprotein-encodinggenesinthecell,andthusallmRNA,iscarriedoutbyRNApolymeraseII.Althoughthisrepresentsasmallpercentageofthetotalmassoftranscriptsproducedinthe

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cell,itmeansthatpolIImusttranscribeanextraordinarilydiversecollectionofgenes.PerhapsforthisreasonthatpolIIhasbeenthefocusofmostofeukaryotictranscription-relatedstudies.Thesestudiesprovidegenetic,biochemical,andcrystallographicdatathatrevealexquisitedetailsaboutthemechanismofthisenzyme.AparticularlyimportantfeatureofthelargestsubunitofRNApolymeraseIIistheC-terminaldomain(CTD)[ 25 ].Thisdomainisanimportantregulatoroftranscriptionelongationandthesubstrateforkeyphosphorylationevents(Section1.5.3). 1.3.3 VacciniaVirusTranscriptioninvacciniavirusiscarriedoutbyamulti-subunitRNA-dependentRNApolymerasethatshareshomologytobothprokaryoticandeukaryoticenzymes[ 26 ].Thishomologyisconnedtothetwolargestsubunits,rpo147andrpo132,RNApolymerasesubunitsof147kDaand132kDa,respectively,andthesmallestsubunit,rpo7[ 27 ].Afourthsubunit,rpo30,isnothomologoustootherpolymerasespersebutdoescontainaregionofhomologywithTFIIS,theeukaryoticanti-arresttranscriptionfactor(Section1.5.2)[ 28 ].Despitethehomologyofrpo147withthelargestsubunitofRNApolymeraseII,rpo147lackstheCTD,implyingthattheregulationofelongationisachievedthroughbindingoffactorstodierentlocationsontheenzyme.TwoformsofthevacciniavirusRNApolymeraseexist:onespecicfortranscriptionofearlygenes,andanotherspecicfortranscriptionofintermediateandlategenescollectively.Structurally,theonlydierencebetweentheseformsisthepresenceofaprotein,theRNApolymerase-associatedproteinof94kDa(RAP94),intheearlyformofthepolymerasethatisabsentintheintermediateandlateform.ThepresenceofRAP94isnecessarybutnotsucientforthespecicityoftheearlyformofthepolymeraseforearlypromoters[ 29 ].

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Regulationofearlygeneexpressiondiersfundamentallyfromregulationofintermediateandlategenes.Earlytranscriptioninvacciniavirusisahighlyregulatedprocess[ 30 ].Promoterrecognitionoccursviaanearlyclass-specictranscriptionfactor[ 31 ].Elongationismediatedbyatleastoneviralelongationfactorwhoseactivitycanbedemonstratedinvitro[ 32 ].Finally,terminationoccurswithhighfrequencyatacis-actingtranscriptionterminationsignal,andismediatedbyatleasttwovirally-encodedtrans-actingfactors[ 32 , 33 , 34 , 35 ].Theearlygeneterminationsignalissimple,ecient,andappearstobeentirelysequence-specic,lackingtheabilitytoformanysecondarystructure.Asatechnicalmatter,earlytranscriptioncanbestudiedusingarelativelypuriedsystem,asvirionssupportthesynthesisofearlyRNAthatispromoter-specic,5'capped,terminatedappropriately,and3'poly-adenylated.Instarkcontrast,theintermediateandlatetranscriptionsystemiseitherunderthecontrolofalessstrictregulatorysystemorunderthecontrolofasystemthatissucientlycomplicatedsoastobeentirelynon-obvious.Furthermore,whileintermediateandlateinitiationoccuratclass-specicpromotersthatarereasonablywelldened,intermediateandlatetranscriptionuseshostproteinsinconjunctionwithviralproteinstoinitiate;complicatingtheidenticationofthecompleteinitiationapparatus[ 36 , 37 ].Elongationandterminationofintermediateandlategenesdierwidelyfromtheelongationandterminationofearlygenes.Therearemultiplefactorsthataectelongation,thoughtheyhavenodenedbiochemicalroleandnodetectablesequencehomologytonon-poxviralelongationfactors.Terminationofintermediateandlategenesisnotresponsivetotheearlygenecis-actingelement.Neitherisitresponsivetoanyotherdetectableterminationsignal;thustranscriptsproducedfromanygivenintermediateorlategenearebothverylongandhighlyheterogeneousinlength[ 38 , 39 ].Furthercomplicatingmattersisthelackofasimpleinvitroassayforintermediateorlategeneelongation,suchas

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theoneavailableforearlytranscription.ThisunfortunatesituationresultsfromthefactthatpuriedvirionsareunabletodirectsynthesisofintermediateorlatemRNA,presumablybecausetheylacktheproperinitiationfactorsforthosegeneclasses.Thisrequirestheuseofinfected-cellextractstodirectintermediateorlatetranscriptioninvitro.Theseextractsareafarmorecomplexmixtureofreagentsthanwhatisfoundinvacciniavirions.Severalclass-specictranscriptionfactorsdirectinitiation,elongation,andterminationofvacciniavirusgenes(Table1-1).Notably,despitethecytoplasmiclocationofviraltranscription,vacciniausesahandfulofhostproteinsinconjunctionwithviralproteinstoregulatetranscription.Transcriptionelongationandterminationofthethreeclassesofvacciniavirusgenesarediscussed(Sections1.5and1.6)inthecontextofelongationandterminationinotherorganisms.Nextisabriefreviewofthethreetranscriptionalstageswithafocusoninitiation,notbecoveredelsewhere. Earlytranscription.Earlytranscriptionoccursinthecontextofthevacciniaviruscoreparticle,andthusiscarriedoutbyaself-containedtranscriptionapparatusthatispackagedwithinvirions.Earlygenesaccountforapproximately50%ofallvacciniagenesandaretranscribeduntilDNAreplicationoccurs.ProductsofearlygenesincludethefactorsneededforDNAreplicationandtranscriptionoftheintermediateclassofgenes.InitiationatearlypromotersreliesontheRAP94subunitofthepolymerase.Alsorequiredforinitiationfromearly(butnotintermediateorlate)promotersistheheterodimericvacciniaearlytranscriptionfactor(VETF),whichisaproductoftheD6RandA7Lgenes[ 40 ].Todate,VETFistheonlypoxviralproteinshowntodirectlybindanyviralpromoter.BindingofVETFtothepromoteroccursatanupstreamelementreferredtoasthe\core"andatadownstreamelement.Thelatterinteractionisapparentlysequence-independent.Onceboundtothe

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promoter,VETFrecruitsthepolymeraseanddissociatesfromthetemplateinastepthatisdependentonitshydrolysisofATP[ 41 ].AnadditionalroleforVETFmaybepackagingoftheearlytranscriptioncomplexonearlyviralpromotersduringmorphogenesis[ 42 ].Inthewild-typevirus,VETFmayserveasanucleationpointforsuchanassembly,asnormalvirionscontainVETF,RNApolymerase,cappingenzyme,andthepoly(A)polymeraselargeandsmallsubunits.ThisideaissupportedbystudiesinwhichsynthesisofeitherVETFsubunitorH4Lareinhibited.H4LmutantspackagenormalamountsofVETF,butfailtopackageRNApolymerase,cappingenzyme,andthepoly(A)polymeraselargesubunit[ 43 ].MutantsinVETFpackagereducedamountsofVETF,RNApolymerase,poly(A)polymerase,NPH-I,andcappingenzyme[ 17 , 42 ]. Intermediatetranscription.TranscriptionofintermediategenesbeginsafterearlytranscriptionandtheinitiationofDNAreplication.Playingtheirpartinthetranscriptionalcascade,intermediategenesencode,amongotherthings,factorsrequiredforlatetranscription.Asinearlytranscription,therecognitionofintermediateandlatepromotersalsorequiresclass-specictranscriptionfactors.Interestingly,thesefactorsareunabletostimulateinitiationoftheearly,RAP94-containingpolymerasefromintermediateorlatepromoters,implyingthatRAP94(inadditiontoactivatingearlytranscription)deactivatesintermediateandlatetranscription.Intermediatemessages,likelatemessages,containanon-templatedpoly(A)headof30-50nucleotides[ 44 ].ThissequenceisthoughttoresultfromaslippagemechanismattheTAAAinitiatorelementthatiscommontointermediateandlatepromoters.Thispropertyofintermediateandlatetranscriptsislikelyafunctionofintermediateandlatepromotersratherthantheintermediateandlatepolymerase,astranscriptionofanearlygenewithaTAAAinitiatorelementalsoresultsinapoly(A)head[ 45 ].Thereisnoknownfunctionorconsequenceofthepoly(A)head

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36 ]identiesG3BPandp137asfactorsthatstimulatetranscriptioninvitrofromthepromoterofanintermediategene,G8R.Whiletheseproteinsco-purifyandformaheterodimer,eachiscapableofstimulatingtranscriptionindependently.BothproteinsarepredictedtobindRNA,andG3BPalsocontainsahelicasedomain,possiblyprovidingacluetoitsmechanism.Althoughthemechanismofactionoftheseproteinsispresentlyunclear,theauthorsproposethattheymaybeinvolvedintransitioninga\slipping"polymerasefrompoly(A)synthesistoanelongation-competentform.TheirstudyalsoaddressedwhetherG3BPorp137activateearlyorlatetranscription,andwhetherothercellularproteinshavesimilaractivity.Finally,itisintriguingtospeculateonwhatadvantagethiscellularactivitymayconferonvirusgrowth.KatsafanasandMosssuggestthatitmayserveasacheckpoint,blockingsubsequentlategeneexpressionuntilG3BPandp137levelsarehigh,somethingthatoccursinanactivelygrowingcell.Whilesuchamodelisappealing(iffornootherreasonthanitpaintsapictureofvacciniavirusaskeenlymanipulatingthehostcellforitsownpurposes),anequallylikelyexplanationisthatthesefactorsroutinelyplayrolesinregulatingcellularRNAsynthesis.Theirinvolvementmaysimplyreecta\crimeofopportunity,"ratherthanaspecicneedoradvantagetocouplingviralmorphogenesiswithcellulargrowth. Latetranscription.Latetranscriptioncompletesthetranscriptionalcascade,synthesizingfactorsrequiredformorphogenesisandtranscriptionofearlygenes

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inthesubsequentlyinfectedcell.Duringmorphogenesis,factorsrequiredforearlytranscriptionarethoughttoassembleontotheearlypromotersfoundinreplicatinggenomesastheyarepackagedintonewlyassemblingvirions.Asaresult,transcriptionofearlygenesinthesubsequentlyinfectedcelloccurswhilestillcontainedinthepartiallyuncoatedcoreparticle[ 46 ].Latetranscriptionrequiresthreeviralinitiationfactors:vaccinialatetranscriptionfactor(VLTF)1,2,and3.Thesefactorswerediscoveredusinganeleganttransfectionassay[ 47 ].Inthisassay,cellsarerstinfectedwithwild-typevacciniavirusinthepresenceofaDNAreplicationinhibitor.InhibitingDNAreplicationblocksthesynthesisofintermediate(andthuslate)genescontainedintheinfectingvirus'genome.Despitetheblockageofintermediatetranscriptionfromtheinfectedgenome,intermediategenescaninfactbeexpressedfromtransfectedDNA.Thisexpressionisrobust,asintermediatetranscriptionfactorsaccumulateduringthetimethatDNAreplicationisblocked.Kecketal.[ 47 ]tookadvantageofthesepropertiesbyco-transfectingareportergeneunderthecontrolofalatepromoterandvarious\test"constructs.Reportergeneexpressionwasobservedonlywhenthecompletesetofintermediate-expressed,latetranscriptionfactorswasco-transfectedalongwiththereporterconstruct.ThisledtotheidenticationofG8R,A1L,andA2Lasgeneswhoseproductstrans-activatelatemRNAexpression.Additionalstudieswererequiredtodeterminewhetherthetrans-activationisdirectorindirect.First,inhibitionofG8synthesisusingarecombinantvacciniaviruscontainingalacoperatorupstreamoftheG8promoterhadnoeectonearlyorintermediatemRNAsynthesis,causedtheaccumulationofintermediateproteins,andinhibitedlategeneexpressiontoapproximately10%ofwild-typelevels[ 48 ].Also,theA1proteinwasshowntofunctionasalate-promoter-specictranscriptionfactorinvitro[ 49 ].Anotherstudy[ 50 ]showedthatA1,A2,andG8producedseparatelyininsectcellsusingabaculovirusexpressionsystemwerenecessaryand

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sucienttocomplementlatetranscriptionactivityofamammaliancellextractmadeinthepresenceofaDNAreplicationinhibitor.Thatstudyshowedthatthereisnodependenceonco-expressionofA1,A2,andG8;andimpliesthatnoneofthethreeworksonlythroughanindirectmechanismtoaectthesynthesisofanother.Anadditionalviralfactor,VLTF-4,isastimulatoroflatetranscriptioninvitro[ 51 ].InthestudythatidentiedG8,A1,andA2aslatetranscriptionfactors,onlyfactorssynthesizedafterDNAreplicationcouldbedetected[ 47 ].Surprisingly,VLTF-4issynthesizedbeforeDNAreplication,thusoriginallyallowingittoescapedetection.Theearlyexpressionofalatetranscriptionfactorisananomalyinthecascadesystemoftranscriptionalcontrol.Interestingly,VLTF-4interactsstronglywiththeintermediateandlatetranscriptionelongationfactorG2bothinvitroandinvivo.Thisinteractionhasbeendetectedbyseveralindependentmeans,includingco-purication,co-immunoprecipitation,andayeast2-hybridassay[ 52 ].Surprisingly,aconditionallethalmutationinthegenethatencodesVLTF-4(H5R)hasnoeectontranscriptioninvivo,butcausesadefectinviralmorphogenesis[ 53 ].Thus,thespecicrolesofVLTF-4invivoarenotclear.However,itsinteractionwithG2andeectontranscriptioninvitrosuggestaroleforthisproteininregulatingelongation,perhapsasanadapterthatlinksG2totheRNApolymeraseortoothertrans-actingelongationfactors.Transcriptionoflategenes,likethatofintermediategenes,isenhancedbycellularproteins.Thosethataectlategenesynthesisincludetheheterogeneousnuclearribonucleoproteins(hnRNPs)A2/B1andRBM3.Generally,thehnRNPsareproteinsthatbindsingle-strandednucleicacidsandplayrolesinRNAmetabolism,splicing,transport,anddecay.WhenpuriedfromanE.coliexpressionsystem,theA2/B1andRBM3proteinsindependentlystimulatelatetranscriptioninaninvitroassay[ 54 ].

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Table1{1:Vacciniavirustranscriptionfactors EarlyVETFD6R,A7LViralPromoterrecognition,ATPaseNPH-ID11LViralElongation,termination,bindingtoRAP94,transcriptreleaseVTFD1R,D12LViralTermination,mRNAcappingenzyme,intermediateinitiationfactor IntermediateVITF-1E4LViralInitiation,rpo30,possiblyrecoveryfromarrestVITF-2G3BP,p137HostInitiationVITF-3A8R,A23RViralInitiationVITF-AD1R,D12LViralInitiation,mRNAcappingenzyme,earlyterminationfactorYY1HostPossiblyinitiation Intermediate/LateA18A18RViralTranscriptreleasefactor,possiblyterminationfactorG2G2RViralElongationfactor,bindstoH5J3J3RViralElongationfactor,mRNA(nucleoside-2'-O-)methyltransferase,poly(A)polymerasestimulatoryfactor LateVLTF-1G8RViralInitiationVLTF-2A1LViralInitiationVLTF-3A2LViralInitiationVLTF-4H5RViralStimulatestranscriptioninvitro,bindstoG2VLTF-XA2/B1,RBM3HostPossibleroleinpromoterrecognition

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1.4 StructureKnowledgeofthestructureofSaccharomycescerevisiaeRNApolymeraseIIandE.coliRNApolymerasehasincreaseddramaticallyduringthelast5years.X-raycrystallographystudieshaverevealeddetailedstructuresfortheseenzymesaloneandincomplexwithDNA,RNA,TFIIS,andtheinhibitor-amanitin[ 55 , 56 , 57 ].WhilenocrystalstructureisavailableforthevacciniavirusRNApolymerase,thestructuralconservationoftheseprokaryoticandeukaryoticenzymesandthestrongdegreeofsequencehomologyinallknownmulti-subunitpolymerases,includingvaccinia,bolsterstheideathatmuchcanbelearnedaboutthestructureofthevacciniaenzymebycomparisonwiththeseotherstructures.Bothprokaryoticandeukaryoticpolymerasesassumeaconformationroughlyresemblingacrabclaw[ 58 ].ThroughthecenteroftheenzymeliesadeepcleftthatislledbythedownstreamduplexDNA[ 57 ].Earlystructuralstudiesindicatedthatthesurfaceoftheenzymeisuniformlynegativelycharged,withtheexceptionofaregioncomprisingthecleftandsaddledomains,providingearlyevidencethattheywereindeedthelocationoftheRNAandDNA[ 59 ].ThestrandsofthisDNAareseparatedatthetranscriptionbubble,aprocessthatisfacilitatedbyvariousloopsofthepolymerasethatmaintaintheopenconformation.Withinthetranscriptionbubble,anumberofforcesareatplaythatstabilizethecomplex.First,themajorityofthetemplatestrandoftheDNAisinvolvedinbasepairinginteractionswiththenascenttranscript.Second,non-pairedtemplatebasesinteractwiththepolymerasebothupstreamanddownstreamoftheheteroduplex.Surprisingly,thenon-templatestrandinteractslittlewiththepolymerase,isnotvisibleinthecrystalstructures,andisthoughttocontributelittletothestabilityofthecomplex[ 57 ]. RNApolymeraseII.Generally,RNApolymeraseIIisconsideredtobea12-subunitenzyme.However,a\core"formcanbeisolatedthatiscomposed

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oftensubunitsandiscapableoftranscriptionelongation,butnotinitiation.Inordertoinitiate,thefourth-largestandseventh-largestsubunits,rpb4andrpb7,arerequiredalongwithanumberofgeneraltranscriptionfactors[ 60 ].Rpb4andrpb7formaheterodimerthatcanassociatewiththecore,andthusthisheterodimeractsasmuchlikeatranscriptioninitiationfactorasitdoesapolymerasesubunit[ 61 ].Regardless,crystalstructuresarenowavailableforboththecoreandthecomplete12-subunitenzyme.Furthermore,comparativestudiesoftheinitiatingandelongatingformsoftheenzymerevealintriguingstructuralchangesthatoccurtoprovidestabilityandthusprocessivitytotheelongatingpolymerase.Functionally,themostinterestingaspectofthepolymeraseisitsprocessivity.ThisenzymeiscapableofsynthesizingRNAmoleculesrequiringcatalyticadditionofthousandsofribonucleotideswithoutdissociationfromtheDNAtemplate.Thisrequirementforstabilityoftheternarycomplexhasresultedinamassiveanduniquepolymerasesub-structurethatlocksthetemplateintoplace.Thisstructureisa50kDaregionofthepolymerasereferredtoastheclamp.OncetheDNAhasboundtothepolymerase,theclampswingsonapeptidehingetosecuretheDNAinaregionknownasthecleft.Thishingeiscomposedofve\switch"regionsthatareexibleandallowresiduesintheclamptomovebyasmuchas14A.Threeoftheveswitchresiduesareincontactwiththeheteroduplex.Thus,theseresiduesmaybethemolecularsensorthatrespondstothepresenceofaDNA/RNAhybridbyinitiatingclosureoftheclamp.Additionalstructuraldomainsalsointeractwiththehybridandarelikelytoplayaroleintheprocessivityofthepolymerase.Ofparticularimportancearepeptideloopsthatextendintothecleftandinteractwithelementsofthetranscriptionbubble,includingthehybrid.Suchloopsincludeforkloop1and2,therudder,thelid,andthezipper[ 56 ].Inmanycases,nodirectrolehas

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beenobservedforthesestructuresintranscription.However,thestructureofthepolymeraseimmediatelysuggestsactivitiesfortheseloopsbasedontheirpositions,andtheconservationofsomeoftheseloopswithbacterialcounterpartsofknownfunctionprovidehypothesestotestdirectly.Forexample,forkloop2isapartiallydisorderedloopthatnonethelesshasanimpliedfunctionbasedbothonitspositionandhomologywithaprokaryoticloop,Dloop1.Thisstructuresitsatthedownstreamedgeofthetranscriptionbubbleandlikelystericallyinhibitsre-annealingoftemplateandnon-templatestrands.Indoingso,forkloop2likelypromotesprocessivitybyinhibitingretrogrademovementofthetranscriptionbubble,andthusthepolymeraseitself.Interestingly,avacciniavirusRNApolymerasemutantmappingtoitshomologousregiondoesdisplayanelongation-defectivephenotypeandisthusconsistentwiththeproposedmechanismofaction[ 62 ].Also,therudderandforkloop1aredisorderedincrystalstructuresoffreepolymerase,butduringtranscription,theycontactoneanotherandcreatespecicpocketsthatmaintainthepositionsoftheRNAanddownstreamDNA[ 63 ].AsolvedstructureprovidescluesaboutfreenucleotideentryandRNAexit.Asmentionedabove,astudyofthesurfacechargedistributionoftheenzymerevealedlikelysitesfortheDNA-andRNA-interactingdomains.However,thecrystalstructureitselfnowmakesobviousothersubstructuressuchastheRNAexitchannel,theporethroughwhichthefreeNTPsenter,andtheNTPbindingsiteandcoordinatingresidues[ 56 , 64 ].Furthermore,thestructuresuggeststhattheNTPpolymerizationandcleavagesitesareoneandthesame[ 65 ].ThesubunitsofpolIIhavehomologsinpolIandpolIIIorarecommontoallthreeenzymes.Withthelargestandsecond-largestsubunitsprovidingthecatalyticactivity,thesmallersubunitslikelyexisttodirectassemblyandprovidestabilitytothecomplex,aswellastoyieldspecicityfortheappropriatepromoterclassandconcomitanttranscriptionfactors[ 56 ].Becauseoftheconservationofthelargest

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twosubunitsbetweeneukaryotic,prokaryotic,andpoxviralRNApolymerases,theemergingdetailsconcerningthedomainsofcellularpolymerasesprovidecluestothefunctionofhomologousregionsinthepoxviralenzymes. 1.5 ElongationTranscriptionelongationistheprocessiveadditionofnucleotidestothe3'endofthenascentRNAtranscript.Likeinitiation,elongationisunderthecontrolofanumberoftrans-actingfactorsthatmayprovideeitherpositiveornegativeregulation.Therateofelongationisalsoinuencedbycis-actingsignalsembeddedinthesequenceitself.Infact,theelongationrateisanextraordinarilydynamicpropertyofthepolymerase,reectingthemanyconformationalstatesthatitmayassumeandthecomplexsymphonyoffactorsthatinuenceit.Muchlikewithtranscriptioninitiation,manyofthestrategiesusedbythesefactorshavebeenconservedacrossphylogeneticboundaries.Forthisreason,webeginwithafocusonE.coliRNApolymeraseandS.cerevisiaeRNApolymeraseII,themostthoroughlystudiedenzymeswithrespecttoelongation.Whereappropriate,theelongationpropertiesofotherenzymeswillalsobediscussed.Finally,thissectionwillculminatewithareviewoftranscriptionelongationfactorsinvacciniavirus.ItisperhapsworthnotingthatitisnotatallclearwhetherpausingcameaboutbecauseofalegitimateneedtoregulatetherateofelongationorasaninherentweaknessinthefunctionofRNApolymerase.Ifthelatteristhecase,thenthemanyfactorsthatexisttorelievepausingwerebornoutoftheneedformaintainingasucientrateoftranscriptproductionbyovercomingtheshortfallsofthepolymerase.Whetherastrengthorweakness,therelationshipbetweenRNApolymeraseandthemanyenzymesthatregulateithasevolvedintoanintricateonethatisessentialtothegoalofproducingRNAoftheappropriatesizeandquantity.

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1.5.1 ElongationMechanismThecurrentmodelofelongationbyanyRNApolymeraseproposesthatthepolymerasemovesalongtheDNAtemplate,addingribonucleotidestothe3'endofthetranscriptusingaratchetingmechanism[ 66 ].Withtheadditionofeachnucleotide,themovementofthepolymeraseisreferredtoastranslocation.Duringelongation,whenatemplatedNTPenterstheactivesite,thepolymeraseformsaphosphodiesterbondbetweenitandthe3'endofthenascentRNA.Atthisstage,thepolymeraseissaidtobeinapre-translocatedstate,meaningthattheactivesiteisoccupiedbythenew3'endoftheRNA.TheenzymemustthenratchetdownstreamonebasepairtofreetheactivesiteandmakeitavailableforentryofthenextNTP.Oncethishastakenplacetheenzymeisinthepost-translocatedstate,andthecyclecontinues.Duringelongation,thepolymerasefrequentlyencountersimpedimentstoitsdownstreammotionwhichcauseittoundergopausing[ 67 ].Theseincludesequence-specicelementsandNTPstarvationinvivo.Pausingcanbeforcedinvitrousingtrans-actingfactorsasroadblocksviabindingtotheDNAdownstreamofthepolymerase.Infact,somemodelsofpausingsuggestthatthepolymerasemaypauseforshortperiodsoftimeateverynucleotideposition.Theimpedimentstoelongationleadtooneoftwoclassesofpausing.ClassIpausinginvolvesformationofanRNAhairpinstructurethatmaintainsthepolymeraseinahyper-translocatedstate,meaningthatthepolymerasehastranslocatedwithoutpriornucleotideaddition.Thus,theactivesiteisincorrectlypositioneddownstreamofthe3'endoftheRNA.Alternatively,classIIpausingoccursduringretrogrademovementofthepolymerasewithrespecttotheDNAtemplate.Thisbacktrackingofthepolymeraseshiftsthepositionofthe3'endofthetranscriptwithrespecttothepolymerase,theDNA-RNAhybrid,andthetranscriptionbubble.Dependingontheextenttowhichthepolymerasehas

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backtracked,thepolymerasecanspontaneouslyrecoverbymovingdownstreamuntilitreachesthepost-translocatedstateorbycatalyzingthecleavageoftheextruded3'endtogenerateanew,appropriatelypositioned3'endintheactivesite.Alternatively,backtrackingcanserveasanintermediatetotranscriptionalarrest.AnarrestedpolymerasehasundergoneaconformationalchangethatcannotbecorrectedwithoutcleavageoftheextrudedRNA3'end.Accessoryfactorsmayparticipateinactivatingthecleavageactivityofthebacktrackedpolymerase,andtheyarerequiredforcleavagebythearrestedpolymerase.CleavageoftheRNAisanevolutionarilyconservedactivityofthepolymerase,andhasbeenobservedinE.coli,S.cerevisiaepolI,II,andIII,andvacciniavirus[ 68 ].TheelongatingRNApolymerasecanassumenumerousconformationalstatesthatdeterminewhetherandatwhatrateitcanelongatethetranscript.Thereareatleasttwostatesinwhichthepolymerasehaselongationactivity,theunactivatedstateandtheactivatedstate.Althoughthepolymeraseisfunctionalintheunactivatedstate,itcatalyzestranscriptionelongationataratemorethanten-foldlowerthanthatoftheactivatedpolymerase.Althoughnotuniversallyagreedupon,apolymeraseintheunactivatedstatemayreachtheactivatedstatebyanallostericregulator[ 69 ].Inthismodel,theregulatorisatemplatedNTPwhichbindsthepolymerasenearthecatalyticsiteoftheenzyme.Thisinteractionmayalterthesuboptimalgeometryoftheactivesite,RNA3'end,andtheDNAtemplatebaseinawaythatrestorestheproperpositioningoftheseelementsandallowsforrapidelongation[ 70 ].Theactivatedstateisrelativelylong-lived,implyingthatthereneednotbeatemplatedNTPpresentintheactivesiteduringeachactofphosphodiesterbondformation.Rather,thepolymeraselikelyenterstheactivestateandremainsinituntillowNTPconcentrationorsequence-specicelementscauseittodecayintotheunactivatedstate[ 71 ].

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Inadditiontoalowerrateofincorporation,theunactivatedstatediersfromtheactivatedstateinitserrorrateandpotentialtoenterother,evenlower-energystates.Forreasonsthatarepoorlyunderstood,theunactivatedpolymerasetranscribeswithanerrorratelowerthanthatoftheactivatedpolymerase.Oneexplanationforthisobservationisthattheunactivatedpolymerasehasthepotentialtoenteralower-energybacktrackedstate.Inthisstate,thepolymerasecanundergofactor-independentcleavageofthe3'endofthenascentRNAtorecovertotheunactivatedstate.Onecircumstanceinwhichcleavagemaybenecessaryismis-incorporationofanon-templatedbase.Inthiscase,thereadyinterconversionoftheunactivatedandbacktrackedstatesprovidesanerror-correctionmechanismthatcouldexplaintheobserveddecreaseinmis-incorporation[ 71 ].Thebacktrackedstateischaracterizedbyextrusionofthe3'endofnascentchainandaninabilitytoperformRNAsynthesis.Thebacktrackedpolymerasecanproceedbackwardsforsomedistance,beforerecoveringtotheunactivatedstatethroughendoribonucleolyticcleavage.Assumingthatthecleavageeventcorrectlypositionsthe3'endofthetranscriptattheactivesiteforelongation,theactivesiteforcleavageactivitymustoccupyasimilarpositiononthepolymerase.Thus,bymeasuringthelengthofthedownstreamcleavageproduct,onecandeterminetheextentofbacktracking.Thismethodhasledtothedeterminationthatapolymerasecanbacktrackasfaras17bases.Abacktrackedpolymerasethatdoesnotundergocleavagemaydecaytothelowest-energystate,arrest[ 72 ].Anarrestedpolymeraseisconsideredtobeinadead-endstatebecauseitisincapableofresumingactiveelongationwithouttranscriptcleavage,eveninthepresenceofhighconcentrationsofNTPs.However,anarrestedenzymeretainstheabilitytobecatalyticallyactive.Itcanberestoredtothelow-energyunactivatedstatethroughcleavageofthetranscript,muchlikewithbacktracked

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enzymes.Wherebacktrackingandarrestdieristhatanarrestedpolymeraserequiresstimulationbyaccessoryfactorsinordertoperformtranscriptcleavage[ 72 , 73 , 74 , 75 ].Awidevarietyofproteinsexistthatpromoteelongationbynumerousmeans.Thesefactorscanbecategorizedaccordingtotheimpedimenttoelongationthattheyworktoovercome.Factorsthatwillbediscussedbelowincludethosethatcounteractpausing,arrest,andtermination.However,thereissomeoverlapbetweengroups,assomeproteinsparticipateinmorethanoneprocessorregulateotherproteinsthatdo.PerhapsthebestcharacterizedelongationfactorsarethosethatinteractwithRNApolymeraseII,andforthatreason,theyarethefocusofthissection.Theimportanceofthesefactorsinvivoisbeyonddispute,asmutationsthatinactivethemareassociatedwithanumberofhumandiseases[ 22 ]. 1.5.2 RecoveryfromArrestInE.coli,theaccessoryfactorsthatpromotecleavageareGreAandGreB,twohighlyrelatedproteinsofapproximately19kilodaltons.WhileGreBhasbeenshowninvitrotostimulateRNApolymerasecleavageactivityatarrestsites,GreAapparentlylacksthatactivity.Rather,itinducescleavagebynon-arrestedpolymerases,perhapspreventingarrestratherthanamelioratingit[ 75 ].Regardless,thefunctionoftheGreproteinsislikelyofsignicantimportance,astheseproteinsarewidelyconservedamongprokaryoticorganisms[ 76 ].Ineukaryotes,theGrehomologTFIISactivatespolIIcleavageactivity.LikeGreB,TFIIScanactoncomplexesafterarresthasoccured.Ithasbeenshowntoactivatecleavagebycomplexesstalledthroughavarietyofmeans.Theseincludearrestsequencesandblockadesbysequence-specicDNAbindingproteins[ 77 , 78 , 79 ].Interestingly,thevacciniavirusRNApolymerasealsohastheabilitytorecoverfromanarrestedstatebyendoribonucleolyticcleavage.Unlikeprokaryoticand

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eukaryoticpolymerases,vacciniapolymeraseappearstoperformthisactivitywithouttherequirementforanactivatorprotein,althoughonevacciniapolymerasesubunit,rpo30,isahomologofTFIIS.Inaninvitrostudy,HaglerandShumandeterminedthatagelpuriedternarycomplexwascompetentforcleavageactivitybyshowingthatthepolymerasecouldremoveanincorporatedchainterminatingGTPanalog,3'-OMeGTP,bycleavageandsubsequentlycontinueelongationofthe5'cleavageproduct[ 68 ].Theseobservationsledtheauthorstosuspectthatthetheactivatormaybeanintegratedcomponentofthepolymeraseitself.BasedonhomologytoTFIIS,suchanactivatorwouldlikelyberpo30.However,nodirectevidenceofaroleforrpo30incleavageactivationexists. 1.5.3 RecoveryfromPausingIncontrasttothefactorsthatfacilitaterecoveryfromarrest,aseparateclassoffactorsworkstopreventarrestbyamelioratingpausing.Thisgroupisrapidlyexpandingwiththediscoveryofnewmembersandcontainsmanyfactorsthatperformwhatappeartobeoverlappingoridenticalfunctions.However,thespecicmechanisticdetailsofhowthesefactorsactisoftenincompletelyunderstood.Rather,whatispresentlyknownaboutmanyofthemistheprocessforwhichtheyarerequired.Thereasonforthatrequirementwillnodoubtbethesubjectofcontinuedexperimentationonthisimportantgroupofenzymes.Duetothelargenumberoffactorsthatcomprisethisgroup,eortwillbemadetofocusonrepresentatives,ratherthanprovidinganexhaustivelistoffactors.TheserepresentativesincludeTFIIF,ELL,Elongin,andP-TEFb,thepositiveregulatorsofelongation,andNELF,anegativeregulatorofelongation.ThepositivetranscriptionelongationfactorsTFIIF,ELL,andElongineecttherateoftranscription.Eachappearstoworkbyreducingtheamountoftimethatthepolymerasespendsinapausedstate,ratherthanbyacceleratingtherateofphosphodiesterbondformation.Thespecicsofthesearediscussedbelow.

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TFIIF.ThepolII-specicgeneraltranscriptionfactorTFIIFplaysaroleinrecruitingthepolymerasetothepromoter,escapefromthepromoter,andsuppressionofpausing.ThisenzymeisaheterodimercomposedofRNApolymerase-associatedproteinsof30kDaand74kDa(RAP30andRAP74,respectively).Bothofthesesubunitshavebeenshowntoplayrolesinbothinitiationandelongation.TheroleofTFIIFinregulatingtheseprocessesismodulatedprimarilybyphosphorylationofRAP74.Toanextent,thisphosphorylationismediatedbyRAP74itself.However,otherkinasesalsoparticipateinthisprocessandareimportantforregulatingfunctionaswell.Thepositionsofphosphorylationhavebeendescribed[ 80 ],ashasthestructureoftheRAP74subunititself[ 81 ].Despiteitsroleinbothinitiationandelongation,TFIIFdoesnotremainassociatedwiththepolymeraseonceithasclearedthepromoter.Rather,itlikelyrecognizesstalledelongationcomplexesandbindsthemtoinduceastructuralrearrangementthatpromotesareturntoactiveelongation[ 82 ].ArecentmechanisticstudyofpolIIelongationinthepresenceofTFIIFconcludesthatthespecicfunctionofTFIIFistostabilizetheelongationcomplexinthepost-translocatedstate[ 83 ]. Elongin.LikeTFIIF,theElonginproteinfamilyisthoughttopromoteelongationbyinteractingwithpausedcomplexesandcausingthemtoresumeelongation.ElonginfunctionsasaheterotrimercomposedofElonginsA,B,andC.ElonginAisthesubunitwithelongationfactoractivity,whileElonginsBandCserveasregulators,thoughtwootherElonginAfamilymembers,A2andA3,werediscoveredthatdonotformcomplexeswithElonginsBandC.Recently,theroleofDrosophilaElonginAwascharacterizedinvivousingRNAinterference[ 84 ].TheauthorsreportthatElonginAisanessentialgenewhoseexpressionpeaksduringthelarvalstageandwhoseproductisrequiredformetamorphosis.Determining

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whetherthehumanhomologhasanimportantdevelopmentalroleawaitsfurtherexperimentation.TheElonginBCcomplexadditionallyservespurposesotherthanregulationofElonginA.Thiscomplexhasbeenshowntointeractwiththesuppressorofcytokinesignaling1(SOCS-1)andthevonHippel-Lindau(VHL)tumorsuppressor[ 85 ].ItislikelythatthefunctionoftheElonginBCcomplexextendswellbeyondregulationofElonginA,SOCS-1,andVHL.InteractionsoftheElonginBCcomplexwithmembersoftheras,WD-40repeatandankyrinrepeatfamilieshavebeendemonstrated[ 86 ].Thus,ElonginAfamilymemberscanfunctionwithoutElonginsBandC,andtheElonginBCcomplexhasfunctionsthatdonotrequireElonginA. ELL.AnotherexampleofanelongationfactorthatactstocounteractpausingisELL,whichperhapsfunctionsviaamechanismsimilartothatofTFIIFandElongin.LikeElonginA,ELLrecentlyhasbeenshowntobeessentialfordevelopmentinDrosophila[ 87 , 88 ].TheessentialnatureofbothElonginAandELLshowsthatalthoughtheymayhavesimilarfunctions,particularlyinvitro,theyareunabletocompensateforoneanotherinvivo.Thus,theyarelikelytosupportelongationofdierentgenes,throughdierentimpediments,orindierentcelltypes.TwoELLhomologs,ELL2andELL3,havebeenidentiedandshowntohaveelongationactivitiessimilartoELL[ 89 , 90 ]. P-TEFb/DSIF/NELF.Thedrug5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole(DRB)resultsintheformationofprematurelyterminatedtranscriptsinvivo[ 91 ].Thiseectisalsoobservedinaninvitrotranscriptionassayusingacrudenuclearextract.However,experimentsdoneusingpartiallypuriedpolymeraseinthisassayshowedittobeinsensitivetotheeectsofDRB.Theseobservationsledtoasearchforfactorsinthecrudecellextractthatcouldbeaddedwiththepuriedpolymerasetotheinvitroassaytorestoresensitivitytothedrug[ 92 ].Thiswork

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uncoveredaDRB-sensitivity-inducingfactor(DSIF)andsubsequenteortsbyothersuncoveredtwoadditionalenzymes,positivetranscriptionelongationfactorb(P-TEFb),andnegativeelongationfactor(NELF)thatworkinconjunctiontoregulatepolIItranscription[ 93 , 94 ].TheP-TEFbheterodimeriscomposedofcyclin-dependentkinase9(cdk9)andcyclinT1,cyclinT2a,cyclinT2b,orcyclinK[ 95 , 96 , 97 ].Thisproteinenhanceselongationbyphosphorylatingtheserine2positionoftheheptadrepeatslocatedontheC-terminaldomain(CTD)ofthepolIIlargestsubunit[ 98 ].PhosphorylationoftheCTDhaslongbeenassociatedwiththetransitionfrominitiation-competentpolIItoaformthatiselongation-competent[ 99 ].IntheabsenceofP-TEFb,abortiveinitiationoccurs,resultingintheproductionofseverelytruncated,non-functionalRNAs.Also,DRBhasbeenshowntoinhibitthekinaseactivityofP-TEFbandhenceitselongationfactoractivityaswell[ 100 ].LikeP-TEFb,DSIFwasidentiedasafactorthatconferredDRBsensitivitytoaninvitrotranscriptionassay.TheheterodimericDSIFconsistsofSpt4andSpt5,twoproteinswithelongationfactoractivity[ 101 ].IthastheabilitytodirectlybindpolIIelongationcomplexes,whereitfunctionstoinhibitpausingandtermination.Additionally,DSIFhasbeenshowntointeractwithSpt6,anelongationregulatorthatmayremodelchromatin,andthenegativeelongationfactorNELF[ 94 , 102 ].TheinteractionbetweenNELFandDSIFisrequiredforNELFfunctionandisdependentuponDSIFbeingincomplexwithpolII.Thus,intheabsenceofNELF,DSIFactsasapositiveregulatoroftranscription.However,inthepresenceofNELF,DSIFactuallyhasanegativeeectsinceitrecruitsNELFtotheelongationcomplex[ 103 ].TheSpt5subunitofDSIFandRDsubunitofNELFaresubstratesforphosphorylationbyP-TEFb.ThesephosphorylationeventsmayresultindissociationofNELFfromDSIF/polIIandtheresumptionofelongation[ 104 , 105 ].

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Interestingly,thisregulatorysystemisexploitedbyhumanimmunodeciencyvirus(HIV)toensuretranscriptionofitsowngenes.TheexpressionofHIVgenesinitiatesfromapromoterwithintheviralLTR,usingthehostpolIItodoso.Thus,HIVdependsonP-TEFbandDSIFtopromoteitsowntranscription.ThevirushasdevelopedanovelmechanismtohijackP-TEFb,relocatingittoHIVpromoter.Thistwo-componentsystemconsistsofacis-actingRNAsequenceelement,thetrans-activationresponseelement(TAR),andtheviraltrans-activator,Tat[ 106 ].Shortlyaftertheinitiationoftranscription,abulge-containinghairpin-loopstructureformsinthenascenttranscript.Thisstructure,theTARelement,isthebindingsiteforTatandisrequiredforTat-dependenttrans-activationofgeneexpression[ 107 ].X-raycrystallographyhasrevealedstructuraldetailsofthisinteractionusingapeptidefragmentofTatthatmaintainsitsabilitytointeractwithTAR[ 108 ].ThemechanismbehindTatactivitybegantoemergewhentheexistenceofaTat-associatedkinase(TAK)wasdemonstrated[ 109 ],andthiskinasewasshowntoberequiredforCTDphosphorylationfromtheHIVpromoter[ 110 ].Additionally,TAKwasshowntoberequiredforformationoflongtranscripts[ 111 ],anditsabilitytophosphorylatetheCTDwasshowntoberesponsibleforTat-dependenttrans-activation[ 112 ].Thetat-associatedkinasewaslateridentiedtobeP-TEFb[ 113 ].RecentexperimentsinaninvitrotranscriptionelongationassayfocusontheroleoftheDSIFsubunitSpt5inTat-dependenttrans-activation.ThisworkshowsthatwhileDSIFisnotrequiredforearlyelongation,itiscriticalforpreventingpolymeraseterminationandarrestatsequencesthatpromotethoseprocessesduringproductiveelongation[ 114 ]. NusAandNusG.TheNusAandNusGproteinsaretwoimportantregulatorsofelongationandterminationinE.coli,andinfacttheyareconservedinallknownbacteria.Theactivitiesofthesetwoproteinsarenotstraightforward

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becausetheyeachexerteectsdirectlyonbothelongationandtermination,ratherthaninuencingoneoftheseprocessesonlyindirectlybyup-regulatingordown-regulatingtheother[ 115 ].ThesedualrolesforNusAandNusGcomplicatetheirplacementinthisdocument.Becausetheycannotbeadequatelydescribedintheabsenceofbackgroundonterminationandanti-termination,adiscussionoftheirfunctionswillbeincludedinthecontextofterminationratherthanelongation. Vacciniaearlyelongation.Despitethethoroughcharacterizationofearlytranscriptioninitiationandtermination,relativelylittleisknownaboutearlyelongation.Currently,onlyoneearlyelongationfactor,nucleosidetriphosphatephosphohydrolaseI(NPH-I)hasbeenidentied.Thisproteinservesdualroles,asitisalsorequiredforearlytermination(Section1.6.6).Usinganinvitrotranscriptionelongationassay,DengandShumanshowthatNPH-IactsasapausesuppressorduringthetranscriptionofaU-richsequenceelement[ 32 ].Additionally,NPH-IisamemberoftheDExHfamilyofATPases.ItsATPaseactivityisssDNAdependentanditselongationfactoractivityapparentlyrequiresitsabilitytohydrolyzeATP,asasingleaminoacidsubstitutionintheATPasedomainabolisheditsabilitytostimulateread-throughofthepausesite.Morerecently,NPH-Ihasbeenshowntointeractwiththeintermediate/lateelongationfactorJ3.AlthoughthisobservationsuggeststheintriguingpossibilitythatNPH-IelongationfactoractivitymightbemediatedthroughitsinteractionwithJ3,invitrostudiesconrmthatthisisinfactnotthecase.DeletionofJ3hasnoeectontherateofearlyelongationinthissystem[ 116 ]. Vacciniaintermediateandlateelongation.Asinbothprokaryoticandeukaryoticsystems,therearepoxviralenzymesthatpromotetranscriptionelongation.Ingeneral,farlessisknownaboutthemechanismofactionoftheseenzymesthantheircounterpartsdescribedabove.However,becausetheRNA

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polymerasesfromallthreesystemsarewellconserved,itislikelythattheactivityoffactorsthatregulatethemisconservedaswell.Thus,onewouldexpecttondfunctionalhomologsofpausesuppressorssuchasP-TEFbandNusGandfunctionalhomologsoffactorsthatpromoterecoveryfromarrest,suchasTFIISandtheGrefactors.Indeed,apoxviralprotein,rpo30,sharessequencesimilaritywithTFIISandmayplayaroleactivatingRNApolymerase-directedendoribonucleolyticcleave.Inothercases,positivetranscriptionelongationfactorsthathavebeenidentiedbyfunctionalassaysbearnosequencesimilaritywithnon-poxviralelongationfactors.Theseelongationfactors,G2andJ3,encodedbytheG2RandJ3Rgenes,respectively,aredescribedbelow.InitialstudiesofG2RrevealedbasicpropertiesofthemRNAandprotein.TheG2Rgeneisexpressedearlyduringinfection,consistentwitharolelateduringinfection.Curiously,theG2RmRNAcontainsa600bp5'untranslatedregion,somethingthatisunusualforvacciniavirustranscripts.Althoughnofunctionhasbeenassignedtothisregion,analysisiscomplicatedbythefactthatanothergene,G3R,residesontheopposingstrand.Thetotaltranscriptlengthisapproximately1.3kb,anditencodesaproteinof26kDa[ 117 ].TheG2Rgeneisessentialforvirusgrowthintissueculture.TheG2proteinwasoriginallyidentiedasapotentialregulatoroftranscriptionwhenitwasdiscoveredthataG2Rmutantvirusisdependentupontheanti-poxviraldrugisatin--thiosemicarbazone(IBT)forgrowth[ 117 ].Isatin--thiosemicarbazone(Section1.6.7)isadrugthatpromotestranscriptionelongation(orinhibitstermination)ofintermediateandlatetranscriptsandcanthereforebeusedtoselectfortranscriptionmutants.Later,anumberofothermutantswereidentiedthatmaptotheG2RgeneandconferIBT-resistanceorIBT-dependence(Figure5-1).Thesemutantsincludeatemperature-sensitive(ts)virus,andseveralengineeredsubstitutionanddeletionmutants[ 117 , 118 ].Theengineered

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substitutionmutantswereconstructedbyalanine-scanningmutagenesis,atechniquethatinvolvesreplacingclusteredgroupsofchargedaminoacidresidueswithalanineinanattempttodisruptprotein-proteininteractionswithoutaectingproteinfolding.ThisworkrevealedthatG2RmutantscandisplayaspectrumofphenotypeswithrespecttogrowthinthepresenceofIBT,includingresistanceanddependencethatcanvaryaccordingtotemperature.AstudyontheeectsofG2invivoprovidedtherstdirectevidencethatthisproteinregulatestranscriptionelongation.Twomutants,Cts56andG2A,theformeratemperature-sensitivevirusfromtheConditcollectionandthelatteranengineeredframeshift,wereanalyzedfortheirproteinandmRNAsynthesisphenotypesinvivo[ 119 ].TheseexperimentsweredoneintheabsenceofIBTandinthecaseofCts56,atthenon-permissivetemperature(40C).Aproteinpulse-labelingexperimentrevealedthatCts56andG2Adisplaynodefectsinearlyproteinsynthesis.However,atlatetimeseachmutantproducesreducedamountsoflargeproteinsrelativetoawild-typecontrolbutnormalamountsofsmallproteins.AnexplanationofthisphenomenonbecameapparentafteranalyzingthepatternofmRNAsynthesisproducedbythesemutants.Bothmutantsproducedearlytranscriptsofwild-typesizeandabundance.However,intermediateandlatetranscriptswerereducedinsize.Thistruncationwasshowntobespecictotheir3'ends,thusdemonstratingthattheG2proteinisrequiredforsynthesisoffulllengthintermediateandlatetranscriptsinvivo.Thetruncatedtranscriptsproducedfromthesemutantsaresucientlylongtoencodesmallintermediateandlateproteinsbutnotlargeones,consistentwiththeproteinsynthesisprole[ 52 ].Interestingly,aG2Rmutationsuppressesmutationofanothervacciniavirusgene,A18R[ 120 ].TheA18Rgeneproduct,aputativeterminationfactor,isdescribedlater(Section1.6.7).

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FollowingthediscoveryoftheG2elongationfactor,anotherproteinwithindistinguishableeectsonelongationwasdiscovered.Thisprotein,J3,isa56kDamultifunctionalenzymeandtheproductoftheJ3Rgene[ 121 ].Inadditiontoitsroleintranscriptionelongation,J3catalyzesRNAprocessingactivitiesatboththe3'and5'endsofthetranscript.Atthe3'end,J3hasbeenshowntofunctionasaprocessivityfactorforthevacciniavirus-encodedpoly(A)polymerase,E1[ 122 ].IntheabsenceofJ3,theE1proteincatalyzestheadditionofapproximately35non-templatedadenylateresiduestothe3'endofnascenttranscripts.However,inthepresenceofJ3,E1extendsthesepoly(A)tractstoatotallengthofapproximately100-150basepairs[ 123 ].Additionally,J3actsasa(nucleoside-2'-O-)-methyltransferase,methylatingthe2'positionofthepenultimatebaseatthe5'endofcappedmessages,andthusconvertingthecap0structureintoacap1structure[ 124 ].ThediscoveryoftheinvolvementofJ3inregulationofintermediateandlategenetranscriptionelongationoccurredinthesamewaysthatithadpreviouslywithG2.SevenspontaneousmutantscapableofgrowthinthepresenceofthedrugIBTweremappedtotheJ3Rgene.Additionally,asuppressorofA18RwasisolatedanditalsomappedtotheJ3Rgene[ 121 ].Collectively,thesetwoobservationsprovidedstrongevidencethatJ3aectstranscriptionelongationinthesamewayasdoesG2.Also,aNorthernanalysisindicatedthatJ3Rmutantvirusesproduce3'-truncatedmRNAs.However,sinceJ3hasatleastthreerolesintranscription,itwasnotimmediatelyclearwhethertheapparentelongationfactoractivitywasinsomewaylinkedtothepoly(A)polymerasestimulatoryandmethyltransferaseactivities.Thisquestionwaselegantlyaddressedusingbothabiochemicalandgeneticsapproach[ 125 , 126 ].WhileJ3hasnumerousactivities,onlyitselongationfactoractivityisessentialforvirusgrowthincellculture.J3Rnullmutantsarecapableofgrowthinthe

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presenceofIBT,stronglyimplyingthatitistheelongationfactoractivityofJ3R,andnotthepoly(A)polymerasestimulatoryor(nucleoside-2'-O-)-methyltransferaseactivitiesthatareessential.AdirectdemonstrationofthiswasshownbyconstructionofaseriesofJ3Rmutantviruses.InthiscollectionofmutantsarethreevirusesthatareeachdefectiveforasingleJ3activityinvivo.Surprisingly,themutantsdefectiveinpoly(A)polymerasestimulationormethyltransferaseactivitiesareeachviable,indicatingthatneitheroftheseactivitiesareessential.Conversely,themutantspecicallydefectiveinelongationwasdependentuponIBTforgrowth,againindicatingtheessentialityoftheelongationactivity.Thesestudiesdeneanelongationdomainonthethree-dimensionalstructureoftheJ3enzyme.Specically,thegeneticmappingofIBT-dependentandIBT-resistantmutantscoupledwiththesolvedX-raycrystalstructureofJ3haveallowedtheidenticationoftwoclustersofresidueswithanessentialroleinelongation.Oneclusterislocatedonthefrontoftheenzyme,nearthemethyltransferaseactivesite,whiletheotherislocatedonthebottomoftheenzyme[ 126 ]. 1.6 TerminationLiketranscriptionelongation,transcriptionterminationschemesareoftenconservedoverwidephylogeneticdistances.ThissectionreviewsterminationofeukaryotesfocusingpolII,E.coliRNApolymerase,andthevacciniavirusearlyandlateRNApolymerases.Acomparisonofthesesystemsrevealsasetofterminationschemesthatdierintheirimplementationbutsharesomebasicactivities.Therearethreeprimarymechanismsfortranscriptionterminationinbacteria:Rho-dependent,intrinsic(Rho-independent),andMutationFrequencyDecline(Mfd)-dependent.TheRho-dependentandRho-independentterminationmechanismsareusedtoterminatethetranscriptionofanRNApolymerasethathasreachedthe3'endofanoperon.Mfd-dependenttermination,onthe

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otherhand,canoccurwhereverapolymerasepauses,forreasonsthatwillbedescribedbelow.ThesemechanismswillbeaddressedinturnbutwiththefocusonMfd,sinceavacciniavirusMfdhomologexistsandmayfunctionbyarelatedmechanism.Anadditionalterminationprocess,attenuation,doesexistinbacteria.Thisisaregulatorystrategy(ratherthanaspecicmechanism)thatusespausingorterminationtocontrolexpressionofdownstreamgenes[ 127 ].Someattenuationmechanismsusetrans-actingfactors,similartoRho-dependenttermination.Othersuseonlycis-actingsequenceelements,similartoRho-independenttermination.Becauseoftheirsimilaritytoothersystemsandtheirpeculiaritytobacteria,theywillnotbediscussedfurtherhere. 1.6.1 IntrinsicTerminationUnlikeRho-dependenttermination,intrinsicterminationrequiresonlythesignalsembeddedwithintheRNAitselftocausetermination.Thesesignalshavetwocomponents,anelementofdyadsymmetrythatformsahairpin-loopstructure,andapoly(U)sequenceapproximately16-20nucleotidesdownstreamofthecenteroftheloop[ 128 ],or7-8basepairsfromthebaseofthestem,regardlessofthestemlength[ 129 ].Kineticandthermodynamicstudiesofthepolymerasesuggestthattheterminationprocessisindirectcompetitionwithelongationateverynucleotideposition[ 130 ].Withthisinmind,anunderstandingofintrinsicterminationnecessarilyincludesthekineticrelationshipofthehairpintoelongationandthechangesinstabilitythatthepoly(U)tractconfersontheDNA/RNAhybridandthusontheternarycomplexasawhole.Thehairpin-loopisgenerallyGC-richandmay,dependingonthemodel,inuencestabilityoftheternarycomplex,translocation,orpausingofthepolymerase.Ontheotherhand,thepoly(U)stretchisthoughttobedirectlyresponsibleforterminationduringthepause[ 129 ].

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ArecentmodelofintrinsicterminationsuggestsforwardtranslocationoftheRNApolymerasecomplexisanecessarycomponentofthenormalterminationpathway[ 131 ].Inthismodel,forwardtranslocationofthepolymeraseisaccompaniedbyrewindingoftheupstreamduplexDNAandunwindingofthedownstreamduplexDNA.Inotherwords,asthepolymerasetranslocates,thetranscriptionbubblemoveswithit.However,becausethereisnonucleotideaddition,thetranscriptisleftbehind,resultinginlossoftheheteroduplexandultimatelyterminationoftheelongationcomplex.Thus,theclosingoftheupstreamedgeofthetranscriptionbubbleunwindstheheteroduplex,resultingintranscriptrelease.Introducingnon-complementarysequencesintotheupstreamportionofthetranscriptionbubbleblocksrewindingofthehelixand,aspredictedbythemodel,resultsinadecreaseintermination[ 131 ].Likewise,theintroductionofacrosslinkintheDNAjustdownstreamofthetranscriptionbubblepreventsstrandseparationduringforwardtranslocationandalsodecreasestheextentoftermination.Terminationcanoccurintheabsenceofdownstreamstrandseparation,howeverthisoccursatamuchlowerrate.AusefultoolininvestigatingthisphenomenonisamutantformoftheEcoRIrestrictionendonuclease,EcoRIE111Q.ThisenzymebindstheEcoRIrecognitionsitebutfailstocleaveit.Thus,itcanbeusedasaroadblocktotranslocationwhenitsrecognitionsiteispositionedatanappropriatedistancedownstreamofanintrinsicterminator.Interestingly,thesestudiesshowthattheroadblockmayincreaseordecreasetheeciencyoftermination,dependinguponitsspacingfromtheterminator.[ 132 ].Nonetheless,theresultscollectivelysuggestthattheprocessofforwardtranslocationofthepolymeraseandtranscriptionbubblearepartoftheinvivoterminationmechanism.AroleofthehairpinstructureinpromotingterminationistounwindtheupstreamportionoftheDNA/RNAhybrid.Theevidenceofthismechanism

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isbasedontheabilitytoperformtranscriptionelongationonebaseatatimeandtheavailabilityofafootprintingmethodthatcandiscriminatebetweentemplateDNAinasingle-strandedordouble-strandedconformation.Thismethod,calledpotassiumpermanganatefootprinting,oxidizesaccessible(non-basepaired)thymidineresiduesbutcannotaccessbasepairedthymidine.Subsequentcleavagewithpiperidinerevealsthepositionsofnon-basepairedthymidines.Thisworkiscarriedoutinaninvitroassayusingasix-histidine-taggedRNApolymerasethatcanbeisolatedfromasolutionbasedonitsanitytonickel-agarosebeads.Usingthissystem,atranscriptionelongationcomplexthatcontainsanintacthairpiniscomparedwithonethatdoesnot.FromthisworkitisclearthattheintacthairpinresultsinstrandseparationoftheupstreamportionoftheDNA/RNAhybrid.Theexactmannerinwhichthisoccursisstillunknown.However,itlikelydependsonthespatialconstraintsassociatedwithformationofdouble-strandedRNAwithintheRNApolymerase[ 133 ].Thedestabilizingeectofthepoly(U)stretchontheternarycomplexmostlikelyresultsfromthefactthatadA-rUbasepairishighlyunstablerelativetoothercombinations[ 134 ].Ahybridrichinsuchpairingprovideslessenergytothestabilizationoftheternarycomplexthandoesanyother.Itlikelyalsoincreasesthelikelihoodthatthehairpincandissociatetheupstreamportionofthehybrid.ThelattersuppositionissupportedbytheobservationthatthethreerUresiduesattheupstreamboundaryofthehybridarethemoststronglyconservedthroughevolution[ 135 ]. 1.6.2 Rho-DependentTerminationIncontrasttotheintrinsicterminator,atrans-actingfactorcalledRhocanalsocauseecienttranscriptiontermination.TerminationsitesspecicforRhoaccountforroughlyhalfofthetotalnumberinE.coli[ 133 ].ThesesiteslacktheG-Crichhairpinstructureandthepoly(U)tractthatarecharacteristicofintrinsic

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terminators.Instead,theRNAneedonlycontainarecognitionsequenceforRho.Thisallowsterminatorstobeplacedatpositionswithinthegenomethatwouldbeincompatiblewiththesequenceorstructureimposedbyintrinsicterminators[ 136 ].Rhoisbothatranslocaseandahelicase.Althoughrelated,thesetwoactivitiesaredistinct.Classically,ahelicaseisanenzymethathastheabilitytoseparatestrandsofaduplex.Varioushelicaseshavespecicityforduplexesofdieringnucleicacidcomposition,meaningthatsomearespecicforDNA/DNA,DNA/RNA,orRNA/RNAhybrids.Othershavemultiplespecicities.Sequencecomparisonsofmultiplehelicasesrevealedconservedsequenceelementsthatareconsideredresponsibleforhelicaseactivity.Logically,thesesequenceelementsweredescribedashelicasedomains.Unfortunately,translocasesareoftenidentiedashelicasesbecausetheycontainthese\helicas"domainswhen,infact,theycontainnodemonstrablehelicaseactivity.FurthercomplicatingtheissuearetranslocasessuchasRhothathavehelicaseactivity.WhileRhocanseparateashort,20-40nucleotideRNA/DNAhybrid,thatactivitymaysimplybeanartifactoftranslocationthatoccursonlyunderinvitroassayconditions[ 137 , 138 ].RhointeractsdirectlywithRNAthroughitstwoRNAbindingdomains.OnedomainisresponsibleforrecognitionandbindingofRhotoaRhoutilizationsite,rut,intheRNA.TherutsiteispoorlyconservedbuttypicallyconsistsofaC-richregionofabout40ribonucleotides[ 139 ].Downstreamoftherutsequence,asecondcis-actingsignalknownasthetranscriptionstoppoint(tsp)existswhereterminationactuallytakesplace[ 140 ].TherutsequenceappearstobemoreimportantthantspforRhospecicity,assubstitutionoftspwithanothersequencestillresultsintermination,althoughatslightlydierentpositions.Infact,terminationwithintspappearstooccurspecicallyatpausesites.Thiscanbeshownbystudyingtheterminationpatternwithinatspsequencedownstreamofarutsequence.Iftherutsequenceisremoved,pausinginthecontextofthetsp

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canbestudied.Suchacomparisonshowsthatterminationwithinthetspsequenceoccursatwhatwouldotherwisebepausesites[ 141 ].TheRhofactorisanassemblyofsixidenticalsubunitsintoaring-likestructure.InitialstructuralstudiesoftheRhofactorwerebasedonreconstructionsofelectronmicrographs.Theserevealedsix-memberedclosedringsandve-memberedopenrings.ThelatterwerepresumedtobetheconformationusedforloadingontoRNA[ 142 ].Morerecently,3.0Aresolutioncrystalstructureshavebeenidentiedofthefull-lengthE.coliRhoproteinincomplexwithsingle-strandedRNAandanATPmimicAMPPNP[ 143 ].Thesearesix-memberedopenringstructures,andthusdierfromeitheroftheelectronmicrographreconstructions.Itnowappearsthatthesix-memberedgappedringisthetrueloadingstructure.Inthisstructure,thereareRNAcontactsintherutsequencewitheachRhosubunit.Uponrutbinding,thegappedringclosesintoanungappedstructureandhydrolyzesATPasittranslocatesuntilencounteringandterminatingapausedpolymerase.ThecanonicalmodelofRho-dependentterminationsuggeststhatRhobindstherutsequenceinthenascentRNAandtranslocatesdownstreaminanATP-dependentfashion.BindingofRhotothetranscriptislimitedbytheco-transcriptionaltranslationofmessagesthatoccursinprokaryotes.Inotherwords,thepresenceofaribosomeonthemessageblocksRhoaccesstoanyrutsitethatisupstreamofatranslationstopcodon.BecausetheribosomesinterferewithRho'saccesstotherutsite,theribosomesblocktranscriptiontermination.Whenarutsiteistranscribeddownstreamofatranslationstopcodon,rutisunprotectedbytheribosomeandavailableforbindingbyRho.ThismeansthatRhotypicallybindsrutquicklyafteritstranscription,ratherthanbindingandscanningtheRNAforastalledpolymeraseoveralongdistance[ 136 ].Uponencounteringastalledpolymerase,Rhoinducesitstermination.TerminationofastalledpolymeraseisthoughttoresultfromRhophysicallypullingthe3'endofthenascenttranscript

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throughtheRNAexitdomainofthepolymerase,thusfreeingtheRNA.ThelossofthestabilizingeectoftheDNA/RNAhybridisthoughttoresultindissociationofthepolymerasefromthetemplate,muchlikewhatisseeninintrinsictermination.Ithasalsobeensuggestedthatintheprocessoftermination,thepolymerasemayenterahypertranslocatedstateaswithintrinsictermination,thoughthereisnoclearevidenceforthisatpresent[ 144 ]. NusAandNusG.TheNusAandNusGproteinseachinteractdirectlywiththeRNApolymeraseandtheterminationfactorRho[ 145 , 146 , 147 ],andeachplaysaroleinananti-terminationprocessmediatedbythebacteriophageproteinN.NusAphysicallyinteractswithN,andNusGinuencesN,atleastindirectlythroughinteractionswithRho[ 147 , 148 ].Insomecases,NusAandNusGhavesimilareectsonRhoandtheRNApolymeraseandtheprocessestheycarryout,howeverinmostcaseseachcounteractsthefunctionoftheother[ 115 ].NusAisa56kDaproteinthatisanegativeregulatorofelongation.NumerousinvitrostudiesprovidedetailsregardingthemechanismofNusAfunction[ 149 ].OnesuchstudyidentiedNusAashavingaroleininuencingpausingattheE.colitryptophan(trp)operonterminationsite.Inthisstudy,NusA-inducedpausingoccursattheterminationsiteandthe1:2hairpinstemstructureofthetrpattenuationsignal.Additionally,NusAwasabletoincreasethepercentageoftranscriptsterminatingattheterminationsitefromapproximately25%tonear100%,thelevelseenduringtrptranscriptioninvivo[ 150 ].MorerecentinvitroassaysatphysiologicalNTPconcentrationyieldsimilarresults.NusAstronglyincreasesdwelltimeatpausesites,inducespausingatsitesnotseeninitsabsence,andslowstherateofelongationby35%[ 151 ].Incontrast,the21kDaNusGproteinisapositiveregulatorofelongation[ 146 ].Perhapscounterintuitively,NusGalsoenhancesRho-dependenttermination[ 152 ].Thesepropertiesareapparentlycontradictory,asonewouldexpectthata

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factorthatdecreasestheextentofpausingwouldalsodecreasetheeciencyofRho-dependenttermination.Whilethisdiscrepancyhasnotbeenentirelyresolved,studieswithaRhomutanthaveshownthattheNusGterminationeectmayinfactberealizedbymodulatingtheinteractionofRhowiththeantiterminationfactorN[ 147 ].Thus,theelongationandterminationpropertiesofNusGmayinfactbetwoseparaterolesforthisenzymeandmaybemediatedbyinteractionswithdierentfactors.AninvitrostudyinvestigatingthefunctionsofNusAandNusGincombinationrevealsthattheyactindependentlyofoneanother[ 151 ].Theauthorssuggestthattheeectsofthetwofactorsarecumulativeandprobablybindthepolymeraseatdierentsitesandwithdierentstoichiometries.AcomparisonofinvitroandinvivotranscriptionratesinthepresenceofbothNusAandNusGsuggeststhattheyalsofunctionindependentlyinthecell.Finally,thepreciseroleofNusAandNusGinelongationandterminationhasyettobedetermined,butkineticstudiesofpausingandterminationinE.colisuggestthatthesefactorsmaybepartiallyresponsibleforstructuralchangesinthepolymerasecomplexthateectitspropensityforterminationataparticularsite[ 140 , 153 ]. 1.6.3 MutationFrequencyDeclineUnlikeintrinsicandRho-dependenttermination,Mfd-dependentterminationisindependentofDNAsequence,perse.Rather,MfdinducesterminationofstalledRNApolymerasethathasencountereddamagetothetemplatestrandduringtranscriptionandrecruitsDNArepairproteins.Thus,Mfdisapartofthetranscription-coupledrepairpathway[ 154 , 155 ].Inordertocarryoutthisfunction,theMfdproteincontainsthreerelevantfunctionaldomains.AttheN-terminusoftheproteinistheUvrBhomologydomain,aregionthatisthoughttobindUvrAandthusrecruitstheDNAexcision-repairmachinerytothestalledtranscriptionelongationcomplex.InthecenteroftheproteinsitstheRNA

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polymeraseinteractingdomain.Finally,nearertheC-terminus,anumberofhelicasemotifsexistwithinatranslocasedomainhomologoustoaregionoftheRecGproteinthoughttopromoteHollidayjunctionmigration.Thesehelicasedomainsmayactasatranslocase\motor"andlikelydonothingtopromoteunwindingoftheDNAstrands[ 156 ].Interestingly,Mfdhasapoxviralhomolog,A18R,thatalsoactsintranscriptrelease. 1.6.4 RibonucleicAcidTracking1TranscriptionterminationfromRNApolymeraseIIiscoupledtomRNAprocessingbyamyriadoffactors.Thesefactorsresultinthecleavageofthetranscriptupontranscriptionofacis-actingRNAsequenceandsubsequentpolyadenylationofthe3'endoftheupstreamcleavageproductthatultimatelyyieldsafunctionalmRNA.Interestingly,thepolymerasecontinuestranscription,furtherelongatingthe3'cleavageproduct.Duetocleavage,mRNAmoleculesproducedfromanygivenpolII-encodedgeneshareacommon3'end.However,thedownstreamcleavageproductsarevariableinlength,possiblyreectingaterminationmechanismthatissequence-independent.Theprocessofelongatingthe3'cleavageproductisrelativelyshort-lived,asthecleavagereactionpromotesthesubsequentterminationofthepolymerase.Thenecessityofthismechanismisreadilyapparent,asapolymerasethatfailstoterminatecaninterferewiththetranscriptionofdownstreamgenesbyblockingaccesstotheirpromotersandalsofailstorecycletoapromoterelementwhereitcaninitiatesynthesisofanothertranscript[ 157 ].OnlyveryrecentlyhavemechanisticdetailsshedlightonthepolIIterminationprocess.ThecurrentlyfavoredmodelsuggeststhatanexonucleaseisresponsibleforterminationofRNApolymeraseII[ 158 ].Thisexonucleaseisaproteinnamedribonucleicacidtracking1(Rat1),afactornamedforitspreviouslydescribedroleinS.cerevisiaemRNAexport[ 159 ].WhencleavageofapolIItranscript

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occurs,thefree5'endofthedownstreamproductisexposed.Accordingtothismodel,Rat1(calledXrn2inhumans)andRat1interactingprotein1(Rai1)arerecruitedtothephosphorylatedCTDofanelongatingpolymerasebytheCTD-bindingproteinRtt103.Upontranscriptcleavage,Rat1\chases"theelongatingpolymerasebyexoribonucleolyticallydegradingthedownstreamcleavageproductuntilitreachesthepolymeraseitself.Thus,thismodelisreferredtoasthetorpedomodel.Thedegradationofthetranscriptisthoughttodestabilizetheternarycomplexandcausetermination.Severallinesofevidencelendcredencetothismodel.First,theRtt103proteinspecicallyassociateswithaCTDpeptidecontainingfourheptadrepeatsthatisphosphorylatedattheserine2residueineachrepeat.Second,tandemanitypuricationofRtt103co-puriesRat1andRai1.Third,chromatinimmunoprecipitationassaysindicatethatallthreeproteinslocalizeprimarilytothe3'endoftranscribinggenes.Fourth,aRat1deletionmutantelongatesbeyondtheterminationpointusedbythewild-typestrain.Finally,apointmutationthattargetsauniversallyconservedresidueinthenucleasedomainofRat1stronglystabilizesthedownstreamcleavageproductproducedatthepolyadenylationsite.Collectively,theseobservationsarguestronglythatRat1isresponsiblefordegradationofthedownstreamcleavageproductandessentialfortermination.TheauthorsdrawareasonableparalleltotheRho-dependentterminationmechanismusedinbacteria(Section1.6.2)inwhichatrans-actingfactorchasesthepolymerasebytraversingthetranscriptandcausingterminationwhenitcollideswiththepolymerase[ 160 ]. 1.6.5 TranscriptionTerminationFactor2Transcriptionterminationfactor2(TTF2)causesRNApolymeraseterminationindependentofanyothertrans-actingfactor.Thisproteinhashelicasedomainsbutnodemonstrablehelicaseactivity,similartoboththeE.coliMfdprotein(Section1.6.3)andthevacciniavirusNPH-Iprotein(Section1.6.6).Additionally,TTF2

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exhibitsdsDNA-dependentATPaseactivityandlikelycouplesthisactivitytotheprocessoftranscriptiontermination.Morebroadly,TTF2isamemberoftheSWI2/SNF2proteinfamily[ 161 ].Membersofthisfamilyperformvariedfunctionsbutgenerallyregulateprotein/DNAinteractionsinthecontextoftranscriptionandDNArepair.Interestingly,TTF2diersfromtheRat1terminator(describedabove),inthatTTF2-dependentterminationisindependentofthephosphorylationstateofthepolymeraseCTD[ 162 ]. 1.6.6 VacciniaVirusEarlyTerminationEarlyterminationisdependentonacis-actingterminationsignalandtwotrans-actingfactors.Thecis-actingsignal,originallyidentiedasTTTTTNTinthenon-templatestrand[ 163 ],waslatershowntoberecognizedasUUUUUNUintheRNAitself[ 33 ].Thissignalisactiveonlyontheearly,RAP94-containingformofthepolymerase,andnotsurprisingly,itisembeddedinmanyintermediateandlateopenreadingframeswithoutconsequence.Theterminatormustbeatleast30nucleotidesupstreamofthe3'endoftheRNA,indicatingthatitisrecognizedonceithasemergedfromtheRNApolymerase[ 164 , 165 ].Additionally,itrequiresboththevacciniaterminationfactor(VTF)andNPH-I[ 32 , 35 , 166 ].Furthermore,theATPaseactivityofNPH-Iisspecicallyrequired[ 32 ].BothVTFandNPH-Iaremultifunctionalenzymes.ThevacciniaterminationfactorservesadditionallyasboththemRNAcappingenzymeandanintermediategeneinitiationfactor,whileNPH-I,inadditiontobeingaterminationfactoralsoservesasanearlygeneelongationfactor(Section1.5.3).Fromthesedata,amodelforearlyterminationhasemerged(Figure1-2).Accordingtothemodel,astheUUUUUNUterminatoremergesfromtheRNApolymerase,itisrecognizedbyVTF.ThistriggersVTFtostimulateNPH-I,causingittohydrolyzeATP,thusprovidingtheneededenergyfortermination.ThissystembearssimilaritytotheRhotranscriptionterminationsystem,as

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Figure1{2:Modelforvacciniavirusearlytranscriptiontermination.Thenucleicacidstrandsaredistinguishedbycolor,withtheDNAtemplatestrandinpurpleandthenon-templatestrandinblue.RedindicatesRNA.TheportionoftheRNAwithintheRNApolymeraseisshownwithadashedborder.Thenon-templatessDNAinthetranscriptionbubbleassociateswithNPH-IandRAP94.Vacciniaterminationfactorrecognizestheearlyterminatorsequence(UUUUUNU)andsignalsNPH-Itoinducetermination.Thediagramandmodelareadaptations[ 167 ].

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bothrequirerecognitionofasequence-specic(notstructure-specic)cis-actingsignal,coupletheexactterminationsitetotherateofelongation,andrequireNTPhydrolysis[ 167 ].AkeydierencebetweentheRhoandNPH-IsystemsisthatRhousesitstranslocaseactivitytocatchupwithandcontactthepolymerase,whileNPH-Iappearstoremainboundtothepolymerasethroughoutproductiveelongation.SinceVTF,notNPH-I,likelyrecognizestheterminatorsignal,thereisnoneedforNPH-Itotranslocate.Finally,theNPH-INTPaseactivityisssDNA-dependent,apropertythatmakesitunlikelytobeabletofunctionasatranslocase,asssDNAiseasilyaccessibleonlyinthecontextofthetranscriptionbubble[ 32 ]. 1.6.7 VacciniaVirusIntermediateandLateTerminationVacciniavirusencodesthe56kDaA18proteinfromtheA18Rgene.LiketheG2Rgene,A18Ralsopossessesalong5'untranslatedregion.Althoughsuchregionsarerareinvacciniavirus,theyplaynoknownfunctionforG2RorA18Rmessages.A18bearssequencehomologyandpossiblefunctionalhomologytotheRhoandMfdtranslocasesandterminationfactors.LikebothRhoandMfd,A18containshelicasedomains.WhilebothRhoandMfdlikelyactastranslocases,RhoisahelicasewhileMfdapparentlyisnot.LikeRho,A18hasbeenshowntohaveaweakhelicaseactivity[ 168 ].ThisactivityeectivelyunwindsshortDNA/DNAhybrids,buthasnoeectonDNA/RNAorRNA/RNAhybrids.Interestingly,theA18helicasedomainsaresimilartothoseofthemammalianERCC3helicase,anenzymeinvolvedinbothtranscriptioninitiationandnucleotideexcisionrepair[ 168 ].Additionally,A18possessesDNA-dependentATPaseactivity[ 169 ].A18isanegativeregulatoroftranscriptionelongation.InterestintheA18proteininitiallystemmedfrommappingtheunusualphenotypeoftemperaturesensitivemutantsCts4,Cts22,andCts23totheA18Rgene.Thisphenotype,designatedabortivelate,isdenedbythesuddenabortionofproteinsynthesis

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lateduringinfection.ThecauseoftheabortivelatephenotypewasshowntobedegradationoftotalRNAandsubsequentlossofproteinsynthesis,presumablyasaresultofthelossofviralmRNAsandthecellularrRNAsandtRNAsrequiredtotranslatethem.Thisphenotypewasshowntoresultfromanincreaseinthelevelofdouble-strandedRNA(dsRNA)inthecell[ 170 ].ThisdsRNAtriggersthelatentdsRNA-activatedribonuclease,ribonucleaseL,andsubsequentlythedegradationoftotalRNA.Interestingly,thisphenotypeisidenticaltothatseenwhenawild-typeinfectionisperformedinthepresenceofIBT.Read-throughtranscriptionofintermediateandlateviralgenescausesincreaseddsRNAlevelsinthecell.StudiestodemonstratethisrstidentiedthattranscriptionofregionsofviralDNAnormallysilentlateduringinfectionarenotsilentduringinfectionwithanA18Rmutant.Thisphenomenonwastermedpromiscuoustranscription.Threehypotheseswereputforthtoexplainpromiscuoustranscription.First,earlypromotersmaybereactivated.Second,thetranscriptionmaybeduetorandom,non-promoter-specicinitiation.Third,read-throughofupstreamlategenescouldberesponsible.NorthernandRT-PCRanalysisofaregionofthevacciniagenomeinwhichalategeneisupstreamofthreeearlygeneswasconducted.Theresultsindicatethatread-throughtranscriptionistoblameforthepromiscuoustranscriptionphenotype[ 171 ].Theobservations(mentionedearlier)thatG2RorJ3RmutationscansuppressamutationinA18Rindicatethattheseproteinsarelikelypartofacommonpathway.BecauseG2andJ3acttopromoteelongation,andA18actstopromotetermination,itbecameclearthatthesethreefactorsarepartofaregulatorysystemthatcontrolsintermediateandlatetranscriptlength.ThedirectinteractionbetweenG2andH5andtheinvitroroleforH5inlatetranscriptionimplicatesthisfactoraswell[ 51 ].

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ToexplorethedirectandindirectrelationshipsthatexistbetweenG2,H5,andA18,Blacketal.measuredinteractionsusingseveraltechniques[ 52 ].Usingco-immunoprecipitation,co-purication,andyeast2-hybridassays,theauthorsdetectedinteractionsbetweeneachpairwisecombinationofproteins.However,someinteractionswerenotdetectedineachassay,andsomerequiredover-expressionoftheproteins.Thus,whilethisstudysupportstheideathattheseproteinsinteractatleastindirectlyaspartofalargercomplex,onlytheH5/G2interactionseemslikelytobedirect.A18isanegativetranscriptionelongationfactorandtranscriptreleasefactorinvitro[ 172 ].ThisactivityisconsistentwithobservationsmadeinvivoofA18Rtemperature-sensitivemutantviruses[ 173 ].SpecicallyA18wasshowntoenhancereleaseoftranscriptsinitiatedattheintermediateG8Rgenepromoter.WhetherA18alsoservesasaterminationfactorwasnotaddressedinthisstudy,butsuchanactivitywouldbeconsistentwiththatofhomologousproteinsinothersystems,suchastheE.coliMfdandRhoproteins. Isatin-thiosemicarbazone.FirstidentiedinasearchforcompoundsthatinhibitMycobacteriumtuberculosis,thethiosemicarbazoneshavesincebeenshowntohaveactivityagainstanumberofviruses.Theseincluderetroviruses,adenoviruses,herpesviruses,arboviruses,reoviruses,andpicornaviruses[ 174 , 175 ].Onesuchcompound,isatin-thiosemicarbazone(IBT,Figure1-3),anditsderivativeswereshowntoinhibitthegrowthofvacciniavirusandotherrelatedpoxviruses.Oneparticularderivativethatshowedactivityagainstvariolavirusinmice,N-methylisatin-thiosemicarbazone(metisazone,methisazone,Figure1-3),wasusedclinicallyintheeorttoeradicatesmallpox[ 176 ].ProducedbyBurroughsWellcomeunderthetradenameMarboran,thiscompoundwastestedforitsabilitytofunctionasbothachemotherapeuticandachemoprophylactic.

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Figure1{3:Isatin-thiosemicarbazone(IBT)andtwoderivativeswithanti-poxviralactivity.ThenumberingoftheisatinandthiosemicarbazoneatomsisshownontheIBTstructureforreference.Primednumbersrefertothethiosemicarbazonechain.Seetheaccompanyingtextfordetails.

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Metisazonehasanegligiblechemotherapeuticbenet.Despitemanystudiesthathighlighttheantiviraleectofmetisazonebothincellcultureandinanimalmodels,humantrialsweredisappointing.Thesetrials,heldinMadras,India,weresummarizedbyBauer[ 175 ].Atbest,metisazoneslightlylowerstheriskofdeathinvaccinatedpersonsexposedtosmallpox.However,itdoesnotlowertheriskofdeathoraltertheseverityofdiseaseforunvaccinatedpatientstreatedwithinvedaysofexposure[ 177 ].Despitethefailureofmetisazoneasachemotherapeutic,itdidoermildbenetsintrialsaddressingitsabilitytofunctionasachemoprophylacticagent.Anearlystudyshowedasignicantbenetfromtreatingindividualsexposedtosmallpox.Inthisstudy,theprobabilityofdiseasewasapproximately25-foldlowerintreatedversusuntreatedindividuals.Furthermore,thenumberofdeathsdecreasedve-fold[ 178 ].However,thistrialhadnumerousdeciencies.Theseincludedthelackofplaceboforthecontrolgroup,failuretorandomizethetreatedandcontrolgroups,andinsucientmonitoringofcomplianceintakingthedrug.Thislastpointisamajorissue,asmetisazonehasbeenrepeatedlyshowntohaveasside-eectsseverenauseaandvomiting[ 177 ].Subsequentstudiesconrmedamodestbenetforprophylacticadministrationofmetisazone.Collectively,theyindicateareductionofdiseaseincidenceofabouttwo-foldandasimilarreductioninriskofdeath[ 177 , 179 , 180 , 181 ].Despiteitsmodestbenet,thereislittlejusticationformetisazoneinlightofthenauseaandvomitingandsubsequentnon-complianceitinducesandthesignicantlygreaterprotectionthatisoeredbyvaccination.Althoughclinicallyirrelevant,IBTanditsderivativesareusefultoolsforthestudyofpoxvirusesandthemechanismsofdrugresistanceanddependence.ThesimplefactthatIBT-resistantandIBT-dependentmutantscanbeisolatedstronglysuggeststhatthemoleculartargetforIBTisaviralfactor[ 182 ].Two

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importantquestionsthusarise.First,whatelementsoftheIBTstructurearerequiredforfunction?Second,whatisthemoleculartargetforIBT?Manystudieshaveattemptedtoaddresseachofthesequestions.Theyaresummarizedbelow.InordertodeterminewhichcomponentsofIBTareimportantforitsfunction,anumberofderivativesweresynthesizedandassayedfortheirantiviralactivity.Interestingly,whileIBTpossessesantiviralactivityagainstvaccinia,variola,rabbitpox,andcowpoxviruses,ithasnoeectonectromelia,alethalpoxvirusofmice[ 183 , 184 , 185 , 186 ].Thus,IBTderivativeswerealsotestedagainstectromeliatodeterminewhetherpotencyagainsteachoftheaforementionedpoxvirusesbyasinglecompoundwaspossible.Surprisingly,thecompoundisatin-4':4'dimethyl-thiosemicarbazone(Figure1-3)wasfoundtoinhibitgrowthofectromeliavirus,however,ithadlostactivityagainsttheotherpoxviruses[ 187 ].WithoutknowingthetargetforIBTorrelatedcompounds,nodetailedexplanationforthisphenomenonispossible.Additionalcompoundsweresynthesizedandassayedfortheirabilitytoinhibitgrowthofwild-typevacciniavirusandtosupportgrowthofIBT-dependentandIBT-resistantvacciniavirusmutants[ 188 ].Thesestudiesdividecompoundsintothreegroups:thosethatinhibitwild-typevirusandsupportthegrowthofanIBT-dependentmutant,thosethatdonotinhibitwild-typevirusanddonotsupportgrowthofanIBT-dependentmutant,andthosethatinhibitwild-type,IBT-dependent,andIBT-resistantviruses[ 189 ].TherstgroupcontainscompoundsthatlikelyshareamechanismwithIBT.Thesecondgroupcontainscompoundsthatarenonfunctional.Finally,sincethecompoundsinthethirdgroupinhibitallthreeviruses,theylikelyworkbyamechanismthatdiersfromthatofIBT,suchasinhibitionofDNAreplication.Takenintotal,thisworkshowedthatmodicationofthethiosemicarbazoneitselfinactivatedthecompound,while

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modicationstotheisatinoftendidnotinterferewithactivity.Infact,theisatincouldbereplacedwithseveralotheraromaticringstructures.Despitedecadesofresearch,themoleculartargetofIBTisstillunknown.However,severalstudieshaveexploredthemechanismofIBTactionusingwild-type,IBT-resistant,andIBT-dependentmutants[ 189 ].ThesestudiesinvestigatedviralprocessessuchasearlymRNAandproteinsynthesis,DNAreplication,intermediateandlateproteinsynthesis,andmorphogenesis.EarlyreportsobservedthatIBTdoesnotinterferewithearlygenetranscriptionandtranslationorDNAreplicationbutdoescauseadefectinlateproteinsynthesis.AnearlyconsensuswasthatIBTinterfereswithtranslationoflateviralmessages[ 190 ]orsynthesisofthevirusparticle[ 191 ].However,morerecentstudieshaveshownthatthedirecteectofIBTisonintermediateandlatetranscription[ 192 ].Isatin--thiosemicarbazoneinhibitstranscriptionterminationorpromotestranscriptionelongationoftheintermediateandlateclassesofmessages.Ineithercase,theresultistheproductionofexcessivelylongintermediateandlateviralmRNAmolecules.BecauseviraltranscriptionoccursfromgenesonbothDNAstrands,longdsRNAmoleculesareproducedinthepresenceofIBT,aphenotypealsoseenwithmutantsintheviralA18Rgene[ 170 ].TheselongdsRNAmoleculestriggertheactivationofthelatentendoribonuclease,RNaseLwhichdegradesbothviralandcellularRNA,resultingtheabruptabortionofviralproteinsynthesis[ 173 ].However,wild-typevacciniavirusisstillIBT-sensitiveevenduringaninfectionofcellsdecientinRNaseLandanotherknowndsRNA-activatedantiviralprotein,PKR,implyingthatanotherantiviralmechanismisstillfunctioninginthesecells.PerhapseitherdsRNAitselfinhibitstranslationorayet-undiscovereddsRNA-mediatedpathwayisresponsiblefortheblockinproductivevirusformation.Thisquestionremainsunanswered.Finally,sinceA18Rmutationhasthesamephenotypeasgrowthofwild-typevirusinthepresenceof

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IBT,itistemptingtospeculatethatthetargetforIBTisA18.HoweveranA18R,G2RdoublemutantassayedforabilitytogrowinthepresenceofIBTrevealedthatevenintheabsenceoffunctionalA18protein,vacciniavirusisstillsensitivetoIBT[ 120 ].IdenticationofamoleculartargetforIBTmaybeforthcomingfromstructuralstudiesofthiosemicarbazones.Thiosemicarbazoneshaveactivityagainstanumberofmicroorganisms(seeabove)andmanyalsopossessanti-tumoractivity.Furthermore,thethiosemicarbazonesformcomplexeswithmetalions,particularlylead,palladium,copper,andiron.Insomecases,theirbiologicalactivityisgreatlyenhancedbysuchanassociation[ 193 ].Manythiosemicarbazone/metalcomplexeshavebeenshowntointeractwithDNA[ 194 , 195 ].Additionally,othershavebeenshowntoinhibitribonucleotidereductase[ 196 , 197 ]andpossiblytopoisomerase[ 198 ].Thus,possiblemechanismsofIBTactionmightwellincludespecicinhibitionofaparticularprotein(ashasgenerallybeenassumed)butmightalsoincludebindingtoviralDNAandthusinterferingwiththeDNAbindingofaviralterminationfactorsuchasA18.OnedicultyindeterminingaprecisemechanismforIBTisthatitisinactiveonearlytranscriptioncomplexes.Thereareatleastthreepossibleexplanationsforthisphenomenon.ThesimplestexplanationisthatIBTmaynotbeabletoenterthecoreparticlewhereearlytranscriptiontakesplace.Alternatively,IBTmayinteractwithaproteinthatispartoftheintermediateandlatetranscriptionelongationorterminationmachinerybutthathasnoroleinearlytranscription.Finally,IBTmayinteractwithearlycomplexesexactlyasitdoeswithintermediateandlatecomplexes,butitseectmaybemaskedbythecis-actingterminationsignalthatisusedbytheearlyterminationapparatus.InsightsintothefunctionoftheA18,G2,andJ3proteinsresultedinamodelofintermediateandlateelongationandtermination(Figure1-4)[ 167 ].According

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tothemodel,A18actsasaterminationfactorandisstimulatedbybindingtotheexposedssDNAofthenon-templatestrandinthetranscriptionbubble.SuchamechanismissimilartothatdescribedforNPH-Iduringearlytranscription.Alsoshownisahostfactor,acomponentofunknowncompositionwhoseexistenceandputativeroleinfacilitatingA18-mediatedterminationisasubjectofdebate.Isatin--thiosemicarbazoneisthoughttobindasiteontheRNApolymeraseitself.G2andJ3maybindthepolymeraseandstimulateelongation.Finally,theH5proteinisassociatedwithG2,thoughitsdirectroleintranscription,ifany,isnotclear.ArevisedmodelispresentedinSection5.7,whereitcanincorporatetheobservationsreportedinourstudy.

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Figure1{4:Modelforvacciniavirusintermediateandlatetranscriptiontermination.Thenucleicacidstrandsaredistinguishedbycolor,withtheDNAtemplatestrandinpurpleandthenon-templatestrandinblue.RedindicatesRNA.TheportionoftheRNAwithintheRNApolymeraseisshownwithadashedborder.Becauseelongationandterminationofintermediateandlategenesmaybemoreintricatelylinkedthanforearlygenes,theJ3,G2,andH5proteinsareshownhere.TheA18proteinalongwithahostfactorterminatetranscription.TheG2andJ3proteinspromotetranscriptionelongation.Thediagramandmodelareadaptations[ 167 ].

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2.1 CellsandVirusesMethodsusedforthepropagationoftheAfricangreenmonkeykidneycelllineBSC40,growthofvacciniavirusstocks,andplaqueassayswereperformedasdescribedpreviously[ 199 ].TheIBT-resistantmutantIBTr90wasoriginallyreportedinastudythatmappedittoaregionwithintheA24Rgene[ 200 ].Independentlyderived,spontaneousIBT-resistantmutantswereisolatedbyDonaldLatner,exactlyasdescribedforIBT-dependentmutants[ 121 ].Briey,tenwell-isolatedplaqueswerepickedfromaplaqueassayofwild-typevirusintheabsenceofIBTandgrowntocreatestocks.Eachofthesetenwild-typestockswasthenplaqueassayedinthepresenceofIBT.Ineachcase,anumberofplaqueswereobservedduetospontaneousmutation.Tenoftheseplaqueswerepickedfromtheplaqueassayofeachofthetenwild-typestocks,foratotalof100virusescapableofgrowthinthepresenceofIBT.These100viruseswereplaqueassayedtodeterminewhethereachwasIBT-resistantorIBT-dependent.AsingleIBT-resistantmutantoriginatingfromeachindependentstockofwild-typeviruswasselectedforfurtherstudy.ThesevirusesweregiventhenamesDL1-3,DL2-4,DL3-2,DL4-10,DL5-7,DL6-1,DL7-3,DL8-1,DL9-4,andDL10-7.DL10-7requiredanadditionalroundofplaquepurication,andwasthereafterreferredtoasDL10-7.1.PreparationsofIBTweremadefreshandusedatanalconcentrationof45M,asdescribedpreviously[ 192 ]. 2.2 PolymeraseChainReactionIsolationofDNAforuseasapolymerasechainreaction(PCR)templatewasperformedusingtheQiagenDNeasyDNAisolationkitaccordingtothe 58

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manufacturer'sprotocol(Qiagen,SantaClarita,CA)oracytoplasmicDNApuricationprotocol,asdescribed[ 201 ].Measurementofabsorbanceat260nmwasusedtodetermineDNAconcentration.PrimersforPCRweredesignedusingVectorNTIversion8.0software(InvitrogenCorp.,Carlsbad,California).Reactionswereoptimizedforprimerandtemplate,butgenerallyconsistedof400ngtemplateDNA,0.32Mprimers(each),2mMMgCl2,10mMdNTPs(each),and2UDeepVentDNApolymerase(NewEnglandBiolabs,Beverly,MA).ProductsofPCRreactionswerepuriedusingAmiconCentriconspinlters(Millipore,Billerica,MA). 2.3 DNASequencingSequencingofviralDNAwasperformedbyPCRamplifyingtheregionofinterestwithanappropriateupstreamanddownstreamprimerandsubmissionofthePCRproducttotheUniversityofFloridaInterdisciplinaryCenterforBiotechnologyResearch(ICBR).IndividualsequencingreadswereassembledintocontigsusingtheWisconsinpackage,version10.3(AccelrysInc.,SanDiego,CA)orVectorNTIversion8.0. 2.4 MarkerRescueMappingAone-stepmarkerrescuemappingofIBT-resistantvacciniavirusmutantswasperformedasdescribedbyCondit,etal.[ 200 ],withminormodications.Briey,dishesofconuent60mmBSC40cellswereinfectedwithDts38,atemperaturesensitivehelpervirusmappingtotheD5Rgene[ 202 ].Disheswerethenco-transfectedwithgenomicwild-typevacciniavirusDNAanda\test"DNAfragment.RatherthanusingclonedviralDNAforthistransfection,PCRproductsampliedfromtheviralgenomeofeachIBT-resistantmutant(Chapter3)orerror-pronePCRproductsampliedfromtheviralgenomeofthewild-typevirus(Chapter4)wereused.Foruseinpreliminarymappingexperiments,PCRamplicationwasdoneusingalibraryofprimersdesignedandgenerouslydonated

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byBenjaminLuttgeandRichardMoyertocreate40overlapping5kbpproductsthatcollectivelyspanthevacciniagenome[ 203 ].Oncea5kbpPCRproductwasidentiedascontainingthemutation,individualopenreadingframesinthat5kbpregionwereampliedfromgenomicviralDNAandusedinasubsequentmarkerrescueexperimenttomapeachmutanttoanindividualgene. 2.5 RNAIsolationConuent60mmdishesofBSC40cellswereinfectedwithwild-typeormutantvirusatamultiplicityofinfection(MOI)of15andincubatedfor9hours.ThecellswerewashedwithPBScontaining0.01%BSAand10mMMgCl2,andtotalcellularRNAwasthenisolatedusingtheRNeasyRNAisolationkit(Qiagen),accordingtothemanufacturer'sprotocol.Twocolumnelutionswereperformedwith50lRNase-freewatereach.Measurementofabsorbanceat260nmwasusedtodetermineRNAconcentration. 2.6 NorthernBlottingRiboprobesspecicforthe500bpatthe5'endofK2L,A10L,andA18RmRNAsweretranscribedfromPCRproducts.Todothis,transcriptiontemplateswerePCRampliedusingaforwardprimercomplementarywiththe5'endoftheopenreadingframe(ORF)andareverseprimercomplementarytoaregion500bpdownstreamofthe5'endoftheORFandtaggedwiththesequence\CGATTTAGGTGACACTATAGAAGCG"containingthebacteriophageSP6promoter.(Theessentialpromoterregionisinitalics,whiletwo5'nucleotidesandfour3'nucleotideshavebeenaddedtopromoteecienttranscription.)TranscriptionfromPCRproductswasperformedwiththeAmbionMAXIscriptinvitrotranscriptionsystem(Ambion,Inc.,Austin,TX).TheRNAsampleswerecombinedwithRNAsampleloadingbuercontainingethidiumbromidetogiveanalconcentrationof20.6%formamide,376mMformaldehyde,and0.4XMOPSanddenaturedbyheatingto70Cfor10minutes.

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Sampleswerethenloadedontoa1%gelcontaining2.2Mformaldehydeand1XMOPSbuer.Gelswereelectrophoresedat20Vfor16hours,duringwhichtimethe1XMOPSrunningbuerwascontinuouslyrecirculatedusingaperistalticpump.TheRNAwaspartiallyhydrolyzedbysoakingthegelsin0.05NNaOH.Thegelswerethensoakedin20XSSC(transferbuer)priortotransfertoaGeneScreenneutralchargemembrane(PerkinElmer,Boston,MA).Membraneswerepre-hybridizedat55Cinahybridizationoven(LabnetInternational,Inc.,Woodbridge,NJ)foratleast2hoursinbuercontaining50mMTris-HClpH7.5,1MNaCl,50%formamide,1%SDS,0.1%sodiumpyrophosphate,10XDenhardt'sreagent(0.2%BSA,0.2%polyvinylpyrolidone,0.2%Ficoll),10%dextransulfate,and0.1mg/mlsalmonspermDNAdenaturedbyheatingto95Cfor10minutes.Followingpre-hybridization,1x107cpmfreshriboprobewasaddedtoeachblotandincubatedovernightat55C.Blotswerethenquicklywashedoncewith1XSSCcontaining0.1%SDSatroomtemperature,fourtimeswith1XSSCcontaining1%SDSat65C,andexposedtolm. 2.7 HomologyModelingHomologymodelsoftherpo147andrpo132proteinswereconstructedusingClustalW,Swiss-PdbViewer,version3.7,andtheSwiss-Modelhomologymodelingserver(http://swissmodel.expasy.org).Anaminoacidsequencealignmentofrpo147andS.cerevisiaerpb1(ProteinDataBankcodes1i6hAand1nikA)orrpo132andS.cerevisiaerpb2(ProteinDataBankcodes1i6hBand1nikB)wasconstructedusingClustalWandusedbySwiss-PdbViewertogenerateastructuralalignment.ThestructuralalignmentwaseditedbyhandandsubmittedtotheSwissModelasanoptimizerequestforconstructionofahomologymodel.TheSwiss-PdbViewerwasusedtoexportviewsofthemodelasMega-PovscenesthatwererenderedusingPOV-Ray(http://www.pov-ray.org)version3.5forLinux.

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2.8 Error-PronePCRError-pronePCRwasperformedasdescribedaboveforstandardPCR,withthreeexceptions.1.TheThermusAquaticusDNApolymerasewasusedinplaceofDeepVentDNApolymerase.2.ThenalconcentrationofdNTPsinthereactionmixturewasunequal,withATPandGTPataconcentrationof5mMandCTPandTTPataconcentrationof25mM.3.MnCl2wasintroducedintothereactionmixtureatanalconcentrationof2mM.Error-pronePCRmutantswereisolatedbyusingthesePCRproductsinamarkerrescueexperiment(Section2.4).However,insteadofoverlayingtheinfected,co-transfecteddisheswithIBT-containingliquidmedia,theywereoverlaidwithanIBT-containingmedia/agarmixturefromwhichindividualplaquescouldbepickedfollowingasixdayincubation.Suchplaqueswerethensubjectedtoaroundofplaquepuricationbeforestocksweregrown.

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3.1 IntroductionTheregulationofintermediateandlategenetranscriptionelongationandterminationiscomplexandknowntobecoordinatedbyseveralproteins.Factorsknowntoplayaroleinelongationorterminationincludethesecond-largestsubunitoftheRNApolymerase,rpo132.Additionally,thenegativetranscriptionelongationfactorandtranscriptreleasefactorA18andthepositivetranscriptionelongationfactorsG2andJ3areimportantregulators.Also,theH5proteinhasbeenimplicatedasastimulatoryfactorforlatetranscription,andbyvirtueofitsinteractionwithG2itmayalsoplayaregulatoryroleinelongation.Moreover,itisentirelypossiblethattheaforementionedproteinsrepresentanincompletelistofregulators.Otherviral(andperhapscellular)proteinsmaycontributesignicantlytoeitherelongationortermination.TwoattemptstoidentifyanysuchproteinsarethefocusofChapters3and4.TheantipoxviraldrugIBTisavaluabletoolforprobingintermediateandlatetranscriptionelongation.BecauseIBTinterfereswithintermediateandlatetermination(orpromoteselongation),itcanbeusedinageneticselectiontoidentifynewmutantswithtranscriptionelongationdefects.PreviousstudiesthatselectedformutantswiththeabilitytogrowinthepresenceofIBTidentiedIBT-dependentmutantsmappingtoG2RandJ3R,andIBT-resistantmutantsmappingtoG2R,J3R,andA24R(thegeneencodingrpo132).TheselectionofadditionalmutantsusingIBTcouldpotentiallyyieldtwobroadcategoriesof 63

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viruses.Therst,comprisedofmutantsmappingtoG2R,J3R,orA24R,wouldlikelyrepresentnewallelesofthesegenesandwouldpotentiallybeinformativewithrespecttothefunctionofspecicdomainsthatcomprisetheproteinsencodedbythesethreegenes.Thesecond,andperhapsmoreinterestingpotentialcategoryofmutantswouldincludethosethatmaptogenesnotpreviouslyimplicatedintheregulationoftranscriptionelongationortermination.SuchmutantswouldlikelymaptoeithernovelelongationfactorsorRNApolymerasesubunitsotherthanrpo132.Acollectionofeightspontaneous,independentlyisolated,IBT-resistantmutantswasassembledbyDonaldLatner.ThiscollectionalsocontainedtwoadditionalmutantsthatwereoriginallydescribedasIBT-resistantbutwerelaterrevealedtobeIBT-dependentmutants.Ananalysisofthesetenmutants(withafocusontheeightIBT-resistantmutants)wasconductedtogainnewinsightsintotheintermediateandlategeneregulatoryapparatus.Theremainderofthischapterdescribesthegeneticmappingandphenotypiccharacterizationofthemutantsinthiscollection. 3.2 Results 3.2.1 PlaqueAssayInordertovisualizetheabilityofeightoftheIBT-resistantmutantstogrowrelativetooneanotherandwild-typevirusinboththeabsenceandpresenceofIBT,aplaqueassaywasperformed.SinceinthepasttheIBTphenotypehasoccasionallybeenlinkedtotemperature,thisexperimentwasperformedattheoptimaltemperatureforwild-typevacciniavirus,37C,andalsoat31Cand39:7C,conditionsthatarepermissiveandnon-permissive,respectively,fortemperaturesensitivevacciniavirusmutants.Appropriatedilutionsfromthe37Cplaqueassayareshownforeachvirus(Figure3-1).Thewild-typevirusisIBT-sensitiveandformslargeplaques,thoughonlyintheabsenceofIBT.Relative

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Figure3{1:PlaqueassayofIBT-resistantmutantsinthepresenceandabsenceofIBT.Thedisheswereincubatedat37Cfor6dayspriortostaining. towild-type,manyoftheIBT-resistantmutantsdisplaysasmallplaquephenotypeintheabsenceofdrug.ForDL1-3andDL10-7.1,plaquesizeisnotinuencedbythepresenceorabsenceofdrug.EachoftheothervirusesformslargerplaquesintheabsenceofIBTthaninthepresenceofIBT,thoughthesizeofthoseplaquesvariesbyvirus.InthecaseofDL5-7andDL8-1plaquesformedintheabsenceofdrugareequivalentinsizetowild-typeplaques.Thus,theIBT-resistantvirusesrepresentaspectrumofplaquephenotypesanddisplaydrugresistancetovaryingdegrees.Twoadditionalmutants,DL6-1andDL9-4(notshown),werediscoveredtobeIBT-dependentat37C.

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3.2.2 GeneticMappingTodeterminethespecicmutationresponsiblefortheIBT-resistancephenotypeofeachmutant,acombinationofmarkerrescuemappingandDNAsequencingwasperformed.BecauseofthehighlikelihoodthatasubsetofthesemutantswouldmaptogenespreviouslyimplicatedinIBT-resistanceorcontrolofelongation,sequencingoftheA18R,G2R,J3R,andA24RgeneswasperformedwithviralDNAfromeachmutant.Ofthetenmutants,fourcontainedmutationsinG2R(DL5-7,DL6-1,DL8-1,andDL9-4),andonecontainedamutationinbothG2RandA24R(DL2-4).MarkerrescueexperimentswereperformedtotestwhethertheIBT-resistanceintheG2RmutantsandG2R/A24Rdoublemutantdoinfactmaptothesegenes.Oftheremainingvemutants,nonecontainedamutationinA18R,G2R,J3R,orA24R.Thus,thesemutantsmustmaptoanovelIBT-resistancegene.MappingstudieswiththesemutantsweredesignedtouncoverthenovelIBT-resistancegene(s)responsiblefortheirphenotype.BecauseDL2-4,DL5-7,DL6-1,DL8-1,andDL9-4wereeachknownfromsequencingtocontainamutationintheG2R,amarkerrescueexperimentwasperformedtodeterminewhethertheirG2RallelecouldconferIBT-resistancetoco-transfectedwild-typegenomesreactivatedbytheDts38helpervirus(Figure3-2).Negativecontrolsinthisandallothermarkerrescueexperimentspresentedincludeuninfectedcells,cellsinfectedwiththeDts38helpervirusbutnottransfected,andcellsinfectedwithDts38andtransfectedwithwild-typegenomicDNA.TheremainingdishesareinfectedwithDts38andco-transfectedwithgenomicwild-typeDNAandaPCRproductampliedfromtheviralgenomeoftheindicatedIBT-resistantmutant.Inthisexperiment,theG2Rgenefromwild-typeviruswasusedasanadditionalnegativecontrol.Thiswasnecessarybecausetransfectionswiththisgene,regardlessoftheallele,tendtoproduceahighbackground(Section4.1).Asignaloverbackgroundwasobservedforeach

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203 ].Thecentralcoreregionofthegenomeishighlyconservedthroughoutthepoxvirusfamilyandnotsurprisinglycontainsthebulkoftheessentialgenes.Thus,initalrescueswereattemptedwith15PCRproducts(Figure3-3,numbers13to17)thatcomprisethisregion.Intherstsuchexperiment,PCRproducts13to27wereampliedfromtheDL1-3genome.Theseproducts,whentransfectedinamarkerrescueexperiment,indicatedthatthemutantmappedtoa5kbpregioncontainingpartofJ3R,allofJ4RandJ5L,andpartofJ6R(dish\20"inFigure3-4).DuetothepriorsequencingofJ3R,itwasknownthatthismutantdidnotmaptoJ3R.BecauseJ4RandJ6ReachencodeRNApolymerasesubunits(rpo22andrpo147,respectively),thelikelihoodthatthismutantwouldmaptooneofthosegeneswashigh,althoughthe15kDaprotein-encodingJ5Lgenewasalsoapossibility.InordertomapthemutanttoJ3R,J4R,J5L,orJ6R,eachoftheseopenreadingframeswasPCRampliedandtransfectedinasecondmarkerrescueexperiment(Figure3-5)similarindesigntotherstone(Figure3-4).ThisexperimentindicatedthatthismutantmappedtoJ6R,thegeneencodingthelargestsubunitoftheRNApolymerase.Thisresultmarkedthersttimethatrpo147hadbeenimplicatedinIBT-resistanceandimpliedthatrpo147wasanessentialregulatoroftranscriptionelongation,andingconsistentwiththeactivityofitsS.cerevisiaehomolog,rpb1.

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Figure3{2:MarkerrescueofviruseswithmutationsinG2R.TheG2RmutationinvirusesDL5-7,DL6-1,DL8-1,andDL9-4(butnotDL2-4)issucienttoconferresistancetoIBT.AsdescribedinSection2.4,conuentdishesofBSC40cellswereuninfected(\uninfected"),infectedwiththeDts38helpervirus(\untransfected"),orinfectedwithDts38andco-transfectedwithwild-typegenomicDNA(\WTGenome")andtheindicatedPCRproduct.Aftera4dayincubationat37CinthepresenceofIBT,disheswerestained.

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Figure3{3:PrimerpairsusedtoconstructPCRproductsformarkerrescue.ProductsampliedusingalibraryofprimersareshownasbluebarsabovetheregiontheyamplifyonaHindIIIrestrictionmapofthevacciniavirusgenome.Eachproductisapproximately5kbinlength.ThePCRproductnumbersshownhereareusedthroughoutourstudy.

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Figure3{4:InitialmarkerrescuemappingofDL1-3.TheDL1-3IBT-resistancephenotypemapstoa5kbpregioncontainingallorpartofgenesJ3R,J4R,J5L,andJ6R.AsdescribedinSection2.4,conuentdishesofBSC40cellswereinfectedwiththeDts38helpervirusandco-transfectedwithwild-typegenomicDNA(\WTGenome")andtheindicatedPCRproduct.Aftera4dayincubationat37CinthepresenceofIBT,disheswerestained.

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Figure3{5:MarkerrescuemappingofDL1-3totheJ6Rgene.AsdescribedinSection2.4,conuentdishesofBSC40cellswereinfectedwiththeDts38helpervirusandco-transfectedwithwild-typegenomicDNA(\WTGenome")andtheindicatedPCRproduct.Aftera4dayincubationat37CinthepresenceofIBT,disheswerestained.

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MappingofDL3-2wasperformedessentiallyasdescribedforDL1-3,exceptthat40,5kbpPCRproductsspanningthegenomewereusedintheinitialroundofmapping.AlthoughDL3-2didnotproduceasignalasrobustasthatseenwithDL1-3,thegenome-widemarkerrescueindicatedthatDL3-2alsomappedtothe5kbpPCRproductcontainingallorpartofgenesJ3R,J4R,J5L,andJ6R(dish\20"inFigure3-6).AshadbeendonewithDL1-3,genesJ3R,J4R,J5L,andJ6RwerePCRampliedfromtheDL3-2genomeandusedinasubsequentmarkerrescueexperiment(Figure3-7).ThisexperimentindicatedthatDL3-2,likeDL1-3,mappedtotheJ6Rgene.BecausebothDL1-3andDL3-2mappedtotheJ6Rgene,theremainingmutants,DL4-10,DL7-3,andDL10-7.1,wereassayedtodetermineiftheymappedtoJ6Raswell.FirsttheJ6Rgenefromeachofthesethreemutantswassequenced.FromthisitwasdeterminedthatDL4-10andDL7-3arewild-typewithrespecttotheirJ6Rgene.Combinedwiththeinitialsequencingeort,thesedataindicatethatDL4-10andDL7-3mustmaptoageneotherthanA18R,G2R,J3R,A24R,andJ6R.Repeatedattemptstomapeachofthesemutantshavebeenunsuccessful(datanotshown).Ontheotherhand,DL10-7.1wasshownbysequenceanalysistocontainamutationinJ6R.MarkerrescueanalysisindicatedthatIBT-resistanceinthismutantdidinfactmaptoitsJ6RmutationandnotanyothergeneimplicatedinIBT-resistance(Figure3-8).Surprisingly,thegenotypeofthismutantwasidenticaltothatofDL1-3(Table3-1). 3.2.3

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Figure3{6:InitialmarkerrescuemappingofDL3-2.TheDL3-2IBT-resistancephenotypemapstoa5kbpregioncontainingJ3R,J4R,J5L,andJ6R.AsdescribedinSection2.4,conuentdishesofBSC40cellswereinfectedwiththeDts38helpervirusandco-transfectedwithwild-typegenomicDNA(\WTGenome")andtheindicatedPCRproduct.Aftera4dayincubationat37CinthepresenceofIBT,disheswerestained.A.DL3-2mapstoPCRproduct\20."B.Amagnicationofdishes\WTGenome"and\20"forclarity.

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Figure3{7:MarkerrescuemappingofDL3-2totheJ6Rgene.AsdescribedinSection2.4,conuentdishesofBSC40cellswereinfectedwiththeDts38helpervirusandco-transfectedwithwild-typegenomicDNA(\WTGenome")andtheindicatedPCRproduct.Aftera4dayincubationat37CinthepresenceofIBT,disheswerestained.

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Figure3{8:MarkerrescuemappingofDL10-7.1totheJ6Rgene.AsdescribedinSection2.4,conuentdishesofBSC40cellswereinfectedwiththeDts38helpervirusandco-transfectedwithwild-typegenomicDNA(\WTGenome")andtheindicatedPCRproduct.Aftera4dayincubationat37CinthepresenceofIBT,disheswerestained.

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Table3{1:GenotypesofspontaneousIBT-resistantmutants DL1-3J6RA535VDL2-4G2R,A24RD1076G,A159TDL3-2J6RS288YDL4-10unknownDL5-7G2RG97VDL6-1G2RTruncation(codon172)DL7-3unknownDL8-1G2RTruncation(codon184)DL9-4G2RTruncation(codon179)DL10-7.1J6RA535V

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ofthegene.InthepresenceofIBT,wild-typevirustranscriptsreachalengththatissucienttoinducedegradationofviralRNA.IntheabsenceofIBT,transcriptsproducedfromtheIBT-dependentG2Avirusareseverelytruncated.However,inthepresenceofIBT,G2Atranscriptsarerestoredtoafunctionallengththatissimilartowild-typetranscriptssynthesizedintheabsenceofIBT.TranscriptsproducedbyeachIBT-resistantmutantarelongerinthepresenceofIBTthaninitsabsence.InnocasedoesanIBT-resistantmutantproducetranscriptsthataresubjectedtodegradation.BecausetheIBT-resistantmutanttranscriptsarenotdegraded,theyarelikelyshorterinlengththanwild-typetranscriptsproducedinthepresenceofIBT,thoughotherexplanationscannotbeformallyexcluded. 3.2.4

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Subsequently,thepulse-labeledRNAcanbeextendedbyaddingbackATP,CTP,GTP,andvaryingamountsUTPandincubatingthecomplex.Gelelectrophoresisofthesetranscriptsallowsforcomparisonofpausingpatternsamongvariousextracts.Withinthelimitationsoftheassay,resultsoftheinvitrotranscriptionstudies(notshown)supporttheinvivoNorthernblottingdata.TheRNApolymerasefromDL1-3andDL3-2infected-cellextractseachexhibitanincreaseddwelltimeatnaturalpausesites.(DL10-7.1hasnotbeenassayedbutispresumablyidenticaltoDL1-3sincetheyhaveincommontheirIBT-resistanceallele.)Thisindicatesthatthesemutantshavedefectsintranscriptionelongation,consistentwiththeirIBT-resistancephenotypeandresponsetoIBTinvivo.TheG2R/A24Rdoublemutant(DL2-4),twounmappedmutants(DL4-10andDL7-3),andthemutantsmappingsolelytoG2R(DL5-7,DL6-1,DL8-1,andDL9-4)areindistinguishablefromwild-typeinvitro.Takenatfacevalue,thiswouldindicatethatthesemutantshavenoelongationdefects,whichwouldbeinconictwiththeinvivodata.Asimplerexplanation,however,isthattheG2proteinisremovedfromtheelongationassayduringthewashstep,andthustheelongationcomplexfromaG2mutantextractisidenticaltothatfromawild-typeextract. 3.2.5 HomologyModelingofRNAPolymeraseSubunitsAthree-dimensionalviewofaprotein'sstructureoftenprovidesinsightsintoitsfunctionthatcannotbeeasilygainedbyothermeans.Unfortunately,thestructureofthevacciniavirusRNApolymerasehasnotbeendeterminedexperimentally.However,itispossibletouseacomputationalapproachtoconstructathree-dimensionalmodelofaproteinifaknownstructureexistsforahomologousprotein.Typically,inordertohavecondencethatsuchamodelisbiologicallyrelevant,thehomologousproteinsmusthaveatleast20%to25%aminoacididentity.Thevacciniavirusrpo147andrpo132proteinseachshare

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Figure3{9:NorthernanalysisofIBT-resistantmutantRNAwithaK2L-specicriboprobe.ThelengthofK2LtranscriptsproducedfromIBT-resistantmutantsrespondstoIBT.AsdescribedinSection2.6,conuentBSC40cellswereinfectedwiththeindicatedvirusatm.o.i.=15inthepresenceorabsenceofIBTandincubatedat37Cfor9hours.TotalRNAwasisolatedandtransferredtoanylonmembrane.ThemembranewasprobedwithariboprobespecicfortheintermediateK2Lgeneandexposedtolm.

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Figure3{10:NorthernanalysisofIBT-resistantmutantRNAwithanA10L-specicriboprobe.ThelengthofA10LtranscriptsproducedfromIBT-resistantmutantsrespondstoIBT.AsdescribedinSection2.6,conuentBSC40cellswereinfectedwiththeindicatedvirusatm.o.i.=15inthepresenceorabsenceofIBTandincubatedat37Cfor9hours.TotalRNAwasisolatedandtransferredtoanylonmembrane.ThemembranewasprobedwithariboprobespecicforthelateA10Lgeneandexposedtolm.

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Figure3{11:NorthernanalysisofIBT-resistantmutantRNAwithanA18R-specicriboprobe.ThelengthofA18RtranscriptsproducedfromIBT-resistantmutantsrespondstoIBT.AsdescribedinSection2.6,conuentBSC40cellswereinfectedwiththeindicatedvirusatm.o.i.=15inthepresenceorabsenceofIBTandincubatedat37Cfor9hours.TotalRNAwasisolatedandtransferredtoanylonmembrane.Themembranewasprobedwithariboprobespecicfortheearly/lateA18Rgeneandexposedtolm.

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approximately21%aminoacididentitywiththeirS.cerevisiaehomologs,placingthematthethresholdofreliablemodel-building.Amodelofrpo132builtfromastructurefortherpb2-containingS.cerevisiaeelongatingRNApolymeraserevealscluesthatmayexplainthephenotypeoftheIBTr90mutation[ 57 ].ThismodelindicatestheY462HmutationpresentinIBTr90issituatednearthebaseofadisorderedloop.Theloop,knownasforkloop2ineukaryotes,isthoughttoplayaroleinmaintenanceofthedownstreamforkofthetranscriptionbubble[ 62 ].Thus,amutationnearforkloop2mayinuencethepositionoftheloopandthusthedegreetowhichitinterfereswiththereannealingofDNAatthedownstreamedgeofthetranscriptionbubble.Usingtheapproachdescribedabovewithrpo132,amodelofrpo147wasconstructedfromastructurecontainingtheS.cerevisiaerpb1proteininthecontextofanelongationcomplex.Basedonthismodel,theputativepositionsintheRNApolymerasestructureoftheDL1-3andDL10-7.1A535VandDL3-2S228Ymutationscouldbeassigned.ThemodelpredictsthatthemutationinDL3-2isneartheactivesiteoftheenzyme.Meanwhile,theDL1-3andDL10-7.1mutationislikelytobeinaregionoftheenzymeknownaspore1.ThisporeisthesitewherefreeNTPsarethoughttodiuseintotheactivesiteandisalsothesitewherethe3'endoftheRNAisextrudedduringbacktrackingofthepolymerase(Section1.5.1). 3.3 ConclusionsSpontaneousIBT-resistantvacciniavirusmutantshavegenerallyfallenintotwocategories:RNApolymerasesubunitsandelongationfactors.Theformercategorywascomprisedofasinglemutant,IBTr90,thatmappedtothesecond-largestsubunitoftheRNApolymerase,rpo132.ThelattercategoryconsistedofanumberofmutantsmappingtotheelongationfactorsJ3andG2.Thegoaloftheworkpresentedinthischapterwastomapadditionalspontaneous

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Figure3{12:Structuralmodelofthevacciniavirusrpo132proteinwithpositionoftheIBTr90mutationindicated.ThemodelisbasedonhomologywiththeS.cerevisiaeRNApolymeraseIIsubunit,rpb2.ConstructionofthemodelwasperformedasdescribedinSection2.7.ThesubstitutedaminoacidresidueinIBTr90(Y462H)ishighlightedinblue.

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Figure3{13:Structuralmodelofthevacciniavirusrpo147proteinwithpositionsoftheDL1-3/DL10-7.1andDL3-2mutationsindicated.ThemodelisbasedonhomologywiththeS.cerevisiaeRNApolymeraseIIsubunit,rpb1.ConstructionofthemodelwasperformedasdescribedinSection2.7.ThesubstitutedaminoacidresidueinDL1-3andDL10-7.1(A535V)ishighlightedinred.ThesubstitutedaminoacidresidueinDL3-2(S288Y)ishighlightedinblue.(A)Frontview.(B)Topview.

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IBT-resistantmutantsinordertoidentifynovelelongation-defectiveallelesofbothelongationfactorsandRNApolymerasesubunitsandtoidentifyadditionalfactorsthatmaybeinvolvedinregulatingelongation.Newelongation-defectiveallelesofG2Rwereidentiedandcharacterized,andforthersttimeIBT-resistancewasmappedtothegeneencodingthelargestRNApolymerasesubunit,rpo147.CharacterizationoftheJ6RmutantsrevealedthattheywerealsodefectiveintranscriptionelongationandwerethereforenotlikelytoexhibitIBT-resistancebecauseofafailuretobindormetabolizethedrug.Proper3'endformationisabalancebetweenelongationandtermination,a\decision"thatmustbemadebythepolymeraseateverynucleotideposition.Thisbalancecanbeinuencedbyseveralfactors.OntheonehandisA18,encouragingthepolymerasetoterminate.OntheotherhandareG2andJ3,encouragingnucleotideaddition.AdditionofIBTtipsthebalanceinfavorofelongation.Torestorethebalance,therearemanyoptions.1.RemoveIBTfromthebalancebyintroducingamutationintothetargetproteinthatpreventsIBTfrombinding.2.RemoveG2orJ3fromthebalancebyintroducingamutationthatinactivateseitherprotein.3.IntroduceamutationintotheRNApolymerasethatmakesitmoresusceptibletoterminationthanthewild-typeenzyme.Thesepossibilitieswillbediscussedinturnbelow.BecauseinfectioninthepresenceofIBTincreasesthelengthofintermediateandlatetranscripts,itmayfunctionbyinhibitingaterminationfactor.Alikelytargetproteinforsuchamechanismisthetranscriptreleasefactor,A18.Ifthisisthemechanismofactionusedbythedrug,itislikelythatmutationsthatleadtosubstitutionofaminoacidresiduesatthedrugbindingsitewouldconferresistancetoIBT.However,thegrowingcollectionofmappedIBT-resistantmutantsisdevoidofA18Rmutants,suggestingthatiftheydoexist,theyarerare.However,IBTcouldalsoworkbybindingtheRNApolymeraseitselfandstabilizingitinsome

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way.Ifthisisthemechanism,themostplausiblewaythatIBTcouldstabilizethepolymerasewouldbetooccupyasiteontheenzymethatisnecessaryforreceivingaterminationsignalfromatrans-actingfactor.ThemostlikelyexplanationfortheIBTresistancephenotypeofthemutantsinourstudyisthatthemutationsarecompensatory,counteractingtheterminationdefectscausedbyIBTwithmutationsthatinterferewithpropertranscriptionelongation.AllmutantsresistanttoIBTandmappingtoG2RandJ3Rarelikelytofallintothiscategory.ThephenotypicanalysisoftheIBT-resistantG2RmutantsDL5-7andDL8-1clearlyindicatethattheyremainresponsivetotheeectsofIBT,asthelengthofK2L,A18R,andA10Ltranscriptsproducedbybothmutantsissignicantlyincreasedinthepresenceofthedrug.Thus,drug-resistanceinthesemutantsprobablyindicatesthattheirG2proteinshavesomeresidualactivity.Theylikelymaketranscriptsthatareshorterthanwild-typelengthintheabsenceofdrugbutarenonethelesssucientlylongtobetranslatedproperly.Inthepresenceofdrug,theirtranscriptsarelonger,butsincethevirusescangrowunderthiscondition,theirtranscriptsareapparentlynotlongenoughtotriggeranydsRNA-mediatedantiviralpathwaythatwouldresultintheabortionofinfection.DierencesintheaveragelengthofIBT-resistantmutantandwild-typetranscriptsarediculttodistinguishbecauseoftheextraordinarilyheterogenouslengthofthetranscripts.However,itisclearthatthetranscriptsproducedinthecontextofanIBT-resistantmutantinfectionarenotdegradedinthepresenceofIBT,thoughtranscriptsproducedbywild-typevirusaredegraded,implyingadierenceinlength.LikemutationofG2RorJ3R,mutationoftheRNApolymerasecouldinterferewithtranscriptionelongation,andthuscouldworkbycompensatingfortheeectsofIBT.Thehomologymodellendssomesupportforthishypothesis.Mutationsinthepore1regionortheactivesitecouldcertainlyinuenceelongationrate.

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Likewise,theycouldalsorepresentthebindingsiteforIBTonthepolymerasethat,whenaltered,allowsthevirustogrowunimpededinitspresence.However,therearetwoindependentlinesofevidencethatsuggestthatthecompensatorymodelcorrectlydescribesthemechanismofdrugresistanceinDL1-3,DL3-2,andDL10-7.1.First,asdescribedabove,invivodataindicatedthattranscriptionofthesemutantsisinuencedbythepresenceofthedrug.Second,invitrodataindicatedthateachofthesemutantsaredecientintranscriptionelongationrelativetowild-typevirus,similartothephenotypeofIBTr90.Insummary,thisworkextendsthecollectionofvacciniavirusmutantsthatcanbeusedtostudytranscription.AlthoughitoersnormevidencetoindicatetheprecisemechanismbywhichIBTinterfereswithgeneregulation,itsuggestsanumberofpossibilities.ThesepossibilitiesandamodelforIBTaction,transcriptionelongationandterminationwillbediscussedinChapter5.

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4.1 IntroductionBecauseisolationandmappingofspontaneousIBT-resistantmutantstendstoidentifynewallelesofknowntranscriptionalregulators,anewapproachisneededtoidentifyunknownregulators.WhilethestudyofpreviouslyidentiedelongationfactorssuchasG2andJ3willlikelyrewardtheinvestigatorwithnew,detailedinformationabouttherolesofthesefactorsinregulatingelongation,itmayprovidelesshelpinansweringbroaderquestionsaboutthecompositionoftheelongationcomplexandtheidentityofeachfactorthatfunctionstoregulateit.Thestudyoftheintermediateandlateelongationcomplexwouldbenetfromamorecompleterosteroftheproteinsinvolved.IsolatingandmappingspontaneousIBT-resistantvirusestoidentifycomponentsoftheintermediateandlatetranscriptionregulatoryapparatussuersfromaproblemofdiminishingreturns.Inthepast,mostIBT-resistantandallIBT-dependentmutantshavemappedtotheelongationfactorsG2andJ3.ThatG2andJ3aresofrequentlythecauseofIBT-dependenceandIBT-resistancereectsthemyriadwaysinwhichtheseproteinscanbemutatedtogiverisetothesephenotypes.MinorlossesinstabilityoractivityofG2orJ3arethoughttobethecauseofIBT-resistanceinmutantsthatmaptoG2RorJ3R,respectively.Therearenodoubtmanywaysinwhichthesegenescanbealteredtocreatesuchaphenotype.ThispresumptionisborneoutbyacomparisonoftheG2Rmutantallelesthatalreadyexist.MutationsleadingtoIBT-resistancedonotclusterinaparticularregionoftheprotein.Rather,theyexistalongitsentirelength.However, 88

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IBT-dependencecanresultfromamuchgreaternumberofpossiblemutations.ThisisthecasebecauseanymutationthatabolishesG2orJ3proteinstabilityorfunctionresultsinIBT-dependence.Suchmutationsincludepointmutations,in-framedeletions,andframeshiftscausedbyinsertionsordeletions.Thus,whenoneselectsformutantvirusthatcangrowinthepresenceofIBT,themajorityofwhatisfoundareG2RandJ3Rnullmutants.Astrategytocircumventthisproblemisneeded.TobegintoaddressthetaskofidentifyingmutantsalteredintheirresponsetoIBTbutnotmappingtoJ3RandG2R,atechniquewasusedthatallowsanygenetobequeriedastowhetherornotintroducingamutationintoitcanresultinviralgrowthinthepresenceofIBT.Thismethodcombineserror-pronePCR,atechniquethatcanbeusedtointroducesemi-randommutationsintoagivenDNAsequence,withmarkerrescueinthepresenceofIBT.Thus,primersspecicforagivenopenreadingframecanbeusedtointroducerandommutationsintothatgivengeneonly.Thisheterogeneouspopulationofmutantallelesisthenco-transfectedintocellsinamarkerrescueprotocol(Section2.4).Onceageneofinterestisidentied,plaquescanbepickedthatshould,ifsuccessful,containpredominatelyIBT-dependentorIBT-resistantvirusmappingtothetargetedgene. 4.2 Results 4.2.1 ScreeningCandidateGenesError-pronePCRandmarkerrescuewerecombinedtoscreenseveralgenesforpossiblerolesinintermediateandlatetranscriptionelongation.Inthisexperiment,cellswereinfectedwiththeDts38helpervirus,andco-transfectedwithwild-typegenomicDNAandaPCRproductcorrespondingtoanopenreadingframetobetested.ThePCRproductwasgeneratedinbothahigh-delityPCRsystem(control)andanerror-pronePCRsystem(experimental).Apositiveresultinthisexperimentwasindicatedbyahighernumberofplaquesondishestransfected

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withtheerror-pronePCRproductthanoncellstransfectedwiththehigh-delityPCRproductcontainingthesamegene.ThegeneschosenforstudyinthisexperimentwereseveralwithknownrolesintranscriptionorthosepreviouslyshowntohavetheabilitytointeractwithRNA.TheseincludedsevenRNApolymerasesubunits(rpo30,rpo7,rpo22,rpo18,rpo19,rpo35,andrpo132),thedsRNA-bindingprotein(E3L),theintermediatetranscriptioninitiationfactors(VITF-1andVITF-3),thelatetranscriptioninitiationfactors(VLTF-1,VLTF-2,andVLTF-3),thelatetranscriptionstimulatoryfactor(VLTF-4),andtheintermediateandlatetranscriptionelongationfactors(G2andJ3).TheG2andJ3geneswereincludedaspositivecontrols.Thesegeneswereexpectedtoproducethestrongestsignal,becauseanymutationthatinactivatesthemshouldproduceaviruscapableofgrowinginthepresenceofIBT(Section4.1).Furthermore,themappingofIBTr90torpo132indicatedthatitshouldbepossibletoconstructamutantintherpo132-encodinggene,A24R.Preliminaryexperimentsindicatedthattheeciencyofamplicationofsequenceslongerthanabout2kbpwasdecreasedintheerror-pronePCRformat.Forthisreason,A24Rwasdividedintothreeoverlappingsegmentsrangingfrom1.2kbpto1.5kbpinlength,designatedA24R-1,A24R-2,andA24R-3fromthe5'to3'end.Furthermore,apilotexperiment(notshown)indicatedthatthe5'-mostfragmentwastheleastlikelytoproduceasignal.Forthisreasonitwasnotincludedinfurtherexperiments.Asexpected,thestrongestsignaldetectedinthisexperimentwaswiththeG2RorJ3Rgenes.However,aclearsignaloverbackgroundwasalsoobservedwithA24R-2,A24R-3,andrpo19.Weakersignalsoverbackgroundwereobservedwithanumberofothergenes,includingE3L,A8RandA23R(VITF-3),H5R(VLTF-4),A1L(VLTF-2),andA2L(VLTF-3).

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Figure4{1:Ascreenofvacciniavirusgenesfortheirinvolvementinregulationofintermediateandlatetranscription.A.Primerpairs(showninblue)thatcollectivelyamplifytheA24Rgeneinthreeoverlappingfragments.B.Ascreenofvacciniavirusgenesfortheirinvolvementinregulationofintermediateandlatetranscription.ConuentdishesofBSC40cellswereinfectedwithDts38andco-transfectedwithwild-typegenomicDNAandtheindicatedPCRproduct.ControlsareasdescribedinthelegendforFigure3-2.Thetoprowofdishesarecontrols.Theremainingdishesarepairedbyrow.Ineachpairedsetofrows,thetoprowcontainsnegativecontrols,transfectedwiththeindicatedhigh-delityPCRproduct.Thebottomrowcontainsdishestransfectedwiththeindicatederror-pronePCRproduct.Aftera4dayincubationat37CinthepresenceofIBT,disheswerestained.

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4.2.2 SpecicityoftheTargetingApproachTodeterminewhetherplaquesformedonthedishestransfectedwitherror-pronePCRproductsaroseasaresultoferrorsinthosePCRproducts,twoplaqueswerepickedforstudyfromeachoftheG2R,A24R-2,andA24R-3dishesofanagar-overlaidexperimentotherwiseidenticaltotheoneshowninFigure4-1.Thevirusesinthoseplaquesweresubjectedtoaroundofplaquepuricationtoensurethatnocontaminatinghelperviruswaspresentandthatthestocksweregeneticallyhomogeneous.TheviralDNAwasthenextractedandusedasatemplateforPCR.Thegeneorgenefragmenttargetedintheoriginalerror-pronePCRexperiment,eitherG2R,A24R-2,orA24R-3wasPCRampliedinahigh-delitysystemforuseinamarker-rescueexperiment.InthecaseoftheA24R-targetedmutants,theothertwofragmentsoftheA24Rgenewerealsoampliedtoensurethattheydidnotcontainmutations.Asexpected,whentheG2Rgenewastargetedformutagenesis,virusescapableofgrowthinthepresenceofIBTandmappingtotheG2Rgenewereproduced(comparedishes\G2R-R1"and\G2R-D1"with\WTG2R"inFigure4-2).Likewise,whentheA24R-2orA24R-3regionsoftheA24Rgeneweretargetedformutagenesis,virusescapableofgrowthinthepresenceofIBTandmappingtothetargetedregionoftheA24Rgenewereproduced(comparedishes\A24R-R1A24R-2"with\WTA24R-2"and\A24R-R2A24R-3"with\WTA24R-3"). 4.2.3 IsolationofanIBT-ResistantH5RMutantThescreenofseveralvacciniavirusgenesfortheirability(whenmutated)togiverisetoIBT-resistantorIBT-dependentmutantsidentiedanumberofgeneswithweakpositivesignals(Figure4-1).Fromthosegeneswithpositivesignals,threewerechosenforfurtherstudy.Thesethreegenes(A2L,rpo19(A5R),andH5R)werechosenbecausetheyencodeproteinsthatdierfromoneanotherwithrespecttotheirbiochemicalactivitiesandknownrolesintranscription.Ofthe

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Figure4{2:G2RandA24Rmutantsconstructedbyerror-pronePCRmutagenesismaptothetargetedgeneorgenefragment.AsdescribedinSection2.4,conuentdishesofBSC40cellswereinfectedwiththeDts38helpervirusandco-transfectedwithwild-typegenomicDNA(\WTGenome")andtheindicatedPCRproduct.Aftera4dayincubationat37CinthepresenceofIBT,disheswerestained.

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threegenes,H5RisofparticularinterestduetoitsknownassociationwiththeG2elongationfactor.InanattempttoisolateIBT-resistantorIBT-dependentA2L,A5R,andH5Rmutants,error-pronePCRproductsampliedintriplicateandcorrespondingtoeachofthesethreegenesweretransfectedinamarkerrescueprotocolinthepresenceofIBT.EachofthethreeH5RtransfectionsandoneeachoftheA2LandA5Rtransfectionsresultedinacytopathiceect(CPE).OncetheCPEwascomplete,infectedcelllysateswereharvested.Virusesintheselysateswereplaquepuriedandthegenetargetedintheerror-pronePCR(A2L,A5R,orH5R)wasPCRampliedfromtheirgenomes.ThesePCRproductswerebothsequencedandusedinamarkerrescueexperimentdesignedtoshowwhethermutantsarisingfromtransfectionoferror-pronePCRproductsinamarkerrescueprotocoldo,infact,maptothegenetargetedintheerror-pronePCR.OneofthemutantsarisingfromH5R-targetederror-pronePCRexperimentcontainstwonon-silentmutationsinH5R.AmarkerrescueexperimentindicatesthattheabilityofthisvirustogrowinthepresenceofIBTmapstoH5R(Figure4-3). 4.2.4 Error-PronePCRMutantGenotypesToanalyzethegenotypeoftheG2RandA24Rmutants,thetargetedgeneineachmutantwassequenced.SequencingtheG2R-targetedmutantsrevealedasingle,non-silentsubstitutionintheG2Rgeneofonemutantandasilentsubstitutiondownstreamofaframeshiftintheothermutant.Likewise,sequencingtheA24R-targetedmutantsrevealedasingle,non-silentmutationineachvirusintheappropriatefragmentbutnomutationsintheotherfragments(Table4-1).SequencingoftheA2L,A5R,andH5Rgenesinerror-pronePCRtargetedmutantscapableofgrowthinthepresenceofIBTwasconducted.TheA2L-targetedandA5R-targetedmutantseachcontainasilentmutationintheirtargetedgene,whiletwoofthethreeH5R-targetedmutantscontainthewild-typeH5Rsequence

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Figure4{3:Amutantconstructedbyerror-pronePCRmutagenesisoftheH5RgenemapstoH5R.AsdescribedinSection2.4,conuentdishesofBSC40cellswereinfectedwiththeDts38helpervirusandco-transfectedwithwild-typegenomicDNA(\WTGenome")andtheindicatedPCRproduct.Aftera4dayincubationat37CinthepresenceofIBT,disheswerestained.TheA24R-2fragmentsofwild-type(WT)andA24R-R1virusesareincludedasnegativeandpositivecontrols,respectively.

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(datanotshown).However,theremainingH5Rmutant,designatedH5R-1,containstwonon-silentmutationsinH5R(Table4-1). Table4{1:Genotypesoferror-pronePCRmutants DistinguishingbetweenIBT-ResistanceandIBT-DependenceThevirusesisolatedbytheerror-pronePCR/markerrescuestrategyarecapableofgrowthinthepresenceofIBT,butthisobservationdoesnotdistinguishbetweenIBT-resistanceandIBT-dependence.Inordertomakesuchadistinction,aplaqueassaywasdonewitheachmutantplustheIBT-sensitivewild-typeandtheIBT-dependentG2Avirusesascontrols.ThisexperimentrevealsthatbothA24RmutantsareIBT-resistant,whileoneG2RmutantisweaklyIBT-resistantandtheotherisIBT-dependent. 4.2.6 HomologyModelingThehomologymodelofrpo132constructedtoanalyzespontaneousIBT-resistantmutants(Section3.2.5)wasalsousedtoapproximatethelocationoftheA24R-R1andA24R-R2mutationsontheprotein'sstructure.IBTr90andA24R-R1sharetheirsinglepointmutation(Y462H).Thisresidueisthoughttoresideatthebaseofforkloop2,aregionimplicatedinmaintainingtheopenconformationofthedownstreamedgeofthetranscriptionbubble.TheA24R-R2mutationislocatedinadierentstructuraldomainonthemodel.Thisstructure,referredtoasthe\wall,"isahighlypositivelychargeddomainwithalikelyroleinbindingtheDNA/RNAhybrid.Interestingly,the\wall"requiresabendofnearly90inthetranscriptionbubble,andthusisamajordeterminantofthestructureassumedbytheDNA/RNAhybridandtheregionsofssDNAinthetranscriptionbubble.

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Figure4{4:G2R-R1,A24R-R1,A24R-R2,andH5R-R1areIBT-resistant,whileG2R-D1isIBT-dependent.Error-pronePCRmutantsweresubjectedtoaplaqueassayinthepresenceandabsenceofIBTtodeterminewhethereachisIBT-dependentorIBT-resistant.TheIBT-sensitivewild-typeandIBT-dependentG2Avirusesareincludedascontrols.

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Figure4{5:Structuralmodelofthevacciniavirusrpo132proteinwithpositionsofA24R-R1andA24R-R2mutationsindicated.ThemodelisbasedonhomologywiththeS.cerevisiaeRNApolymeraseIIsubunit,rpb2.ConstructionofthemodelwasperformedasdescribedinSection2.7.ThesubstitutedaminoacidresidueinA24R-R1(Y462H)ishighlightedinblue,andthesubstitutedaminoacidresidueinA24R-R2(T884A)ishighlightedinred.

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4.2.7 ConclusionsThetechniquespresentedinthischapterdeneaviablestrategyforextendingthenumberofknowngenesinvolvedinintermediateandlatetranscriptionelongation.ThiswasshownbytheconstructionofIBT-resistantandIBT-dependentmutantswithrandommutationsinthespecicallytargetedgenesG2R,A24R,andH5R.WhiletheG2RandA24Rmutantsserveasaproof-of-concept,theseIBT-resistantmutantsadditionallyshouldproveusefulforthestudyoftheG2RandA24Rgeneproductsthemselves.Also,ascreenofotherpotentiallyinterestinggeneshasuncoveredseveralthatwarrantfurtherstudy.TheseincludethesmallRNApolymerasesubunitA5R(rpo19),theintermediateinitiationfactorsA8RandA23R,thelateinitiationfactorsA1LandA2L,andthedsRNA-bindingproteinE3L.Mostimportantly,amutantmappingtotheH5RgeneandcapableofgrowthinthepresenceofIBThasbeenidentied.Thisresultvalidatestheerror-pronePCR/markerrescueapproachforthescreeningofgenesfortheirinvolvementintranscriptionandprovidesstronggeneticevidencetolinkH5Rtotheintermediateandlatetranscriptionregulatorysystem.AnattempttoisolateIBT-resistantorIBT-dependentmutantsinA2L,A5R,andH5RresultedintheisolationofasinglemutantinH5R.Thismutant,H5R-R1,providesinvivoevidencelinkingtheH5proteintoaroleintranscriptionelongationandsupportsitsinvitrocharacterizationasastimulatoroflatetranscription[ 51 ].TheidenticationofanIBT-resistantorIBT-dependentH5RmutantimmediatelysuggestsanumberofexperimentsaimedatreninganyspecicroleofH5inregulatingtranscription.SuchexperimentsshouldincludetheinvitrotranscriptionassaysandNorthernblotsdescribedpreviously(Chapter3).WeretheH5Rmutanttoexhibitnodefectintranscriptionelongation,itwouldsuggestthatH5maybethetargetofIBT.AlsoofparticularinterestisthequestionofwhetherthemutantH5proteinretainsitsabilitytointeractwith

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G2.Todate,nofunctionalsignicancehasbeenassignedtothisinteraction.H5R-R1providesauniqueopportunitytoprobethisinteraction.CertainlythediscoveryofanH5mutantwithaphenotypepreviouslyassociatedwithG2Rmutants(IBT-resistanceorIBT-dependence)increasesthelikelihoodthattheG2/H5interactionisimportantforthefunctionofG2.DeterminingwhetherthisinteractionisessentialforG2functionmayimplicateH5asanactivatorofG2.Interestingly,theIBTr90andA24R-R1mutantssharethesamegenotype.Eachhasatyrosinetovalinesubstitutionatposition462.ThissituationissimilartothatobservedwiththespontaneousmutantsDL1-3andDL10-7.1,whichareanidenticalpairofrpo147mutants(Section3.2.2).Theseobservationssuggestthattheresiduesinvolvedinthesemutationsmaybecriticallyimportantforregulatingelongation.TheyalsosuggestthattheremayarelativelysmallnumberofpossiblepolymerasemutationsthatcanleadtoIBT-resistance.Furtherstudyofthesemutantsmayshedlightonthesematters.

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5.1 IntroductionThemappingandphenotypiccharacterizationofseveralisatin--thiosemicarbazone(IBT)-resistantvacciniavirusmutantshasbeenpresented.Collectively,thesemutantsreneandextendthebodyofknowledgesurroundingtheelongationandterminationpropertiesofthevacciniavirusintermediateandlategenetranscriptionmachinery.Additionally,thisworkprovidesdatathatbeginstoaddressquestionsabouttheanti-poxviralmechanismofactionofIBT.Aplausiblemodeltoexplainintermediateandlatevacciniavirustranscriptionelongationandterminationisconstrainedbyanumberofrelevantobservations.First,invivoandinvitrodataindicatethattheA18RgeneproductfunctionstoopposetranscriptionelongationandtosupportreleaseoftranscriptsfromstalledRNApolymerasecomplexes.AdditionalstudieshaverevealedthatA18possessesATPaseandhelicaseactivities.Second,geneticevidencehasrevealedanessentialroleforboththeG2RandJ3Rgeneproductsinstimulatingtranscriptionelongation.Mutationalinactivationofeitheroftheseproteinsresultsintheproductionofshorter-than-wild-typelengthintermediateandlatetranscripts.Also,theG2proteininteractsstronglywiththeproductoftheH5Rgene,aproteinalreadyimplicatedinlatetranscription.Third,geneticandbiochemicallinesofevidencehaverevealedthatmutationofthelargestandsecond-largestsubunitofthevacciniavirusRNApolymerasecanresultinadecreasedtranscriptionelongationphenotype.Presumably,mutantswithincreasedelongationpotentialarealsoplausible.However,sincethereisnosimplegeneticselectionwithwhichtoidentifythem,nosuchmutantshavebeenidentied.(The 101

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scarcityofRNApolymerasemutantswithanincreasedtranscriptionelongationphenotypethereforeimpliesnothingabouttherelativefrequencywithwhichthesemutantscouldtheoreticallyarise.)Anadditionalobservationthatmayinuenceanelongation/terminationmodelisthatIBTcansubstituteforeithertheG2orJ3protein,butG2andJ3cannotsubstituteforoneanother.Thatis,bothG2andJ3arerequiredintheabsenceofIBT.Finally,therelationshipbetweenRNApolymerase,A18,G2,J3,H5,andIBTisunclear.Ofthese,onlyG2andH5havebeenshowntointeractdirectly,whileA18maycomplexwithG2andH5indirectly.AnobviouscandidateforamediatorofindirectinteractionsamongA18,G2,andH5isthetranscriptionelongationcomplex,however,suchaninteractionhasnotbeendemonstratedexperimentally.Furthermore,interactionsbetweenJ3andtheA18,G2,andH5proteinshavenotbeenthoroughlycharacterized.Agenome-wideyeast-2-hybridscanofthevacciniavirusgenomerevealednodirectinteractionsbetweenJ3andA18,G2,orH5.However,thisisanegativeresultandthusshouldnotbeover-emphasized.Eachoftheseconsiderationsisaddressedinadiscussionbelowthatculminatesinthepresentationofaviablemodel. 5.2 A18TheA18proteinsharesconsiderablesequencehomologyandbiochemicalactivitywithterminationfactorsinbothprokaryoticandeukaryoticsystems,andmayfunctionviaasimilarmechanism.LiketheprokaryoticMfdandRhoproteinsandtheeukaryoticTTF2protein,A18possesseshelicasedomains.HypothesesaboutthemechanismbywhichMfd,Rho,andTTF2induceterminationincludetranslocationalongnascentRNAuntilastalledpolymeraseisreached,followedbyreleaseofthepolymerasefromtheRNAandtemplateDNA.A18likelyfunctionsbyasimilarmechanism.Also,A18maycauseterminationinasequence-independentfashion,similartoMfdandTTF2,explainingtheextraordinaryheterogeneityinthelengthofvacciniavirusintermediateorlate

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RNAsproducedfromanygivengene.Interestingly,Mfdisthoughttoplayaroleintranscription-coupledDNArepairbyrecruitingtherepairmachinerytoanRNApolymerasestalledatthesiteofDNAdamage.ThoughA18hasnotbeenshowntoplayaroleinDNArepair,thisremainsanintriguingpossibility.Manypoxvirusesdoencodeproteinsthatoerresistancetoultravioletlight(UV)-inducedDNAdamage,suchasthecyclobutanepyrimidinedimerphotolyasesencodedbyLeporipoxviruses[ 204 ].ExperimentsmeasuringviralUVsensitivityshowthatmutationoftheDNAgyraseincreasesUVsensitivity[ 205 ],whilemutationofthesecond-largestsubunitoftheRNApolymerase(rpo132)orG2decreasesUVsensitivity(NissinMoussatche,personalcommunication). 5.3 G2andJ3UnlikeA18,themechanismsofactionofG2andJ3arecompletelyobscure.TherehavebeennohomologsofG2identiedoutsideofthepoxvirusfamily,andJ3homologywithnon-poxviralproteinsislimitedtothe(nucleoside-2'-O-)-methyltransferasedomain.Thus,therearefewcluesaboutthemechanismbywhichtheseproteinseecttranscriptionelongation.Thegenetics-basedapproachdescribedbothhereandelsewherehasprovidednewG2mutantswhosecharacterizationisinformativewithrespecttotheimportanceofvariousdomainswithintheG2protein.ThecompletecollectionofG2mutantsrevealsthatmutationsthatconferIBTresistancetotheviruscanbeconstructedinpositionsalongvirtuallytheentirelengthoftheG2protein(Figure5-1).WepresumeforthreereasonsthatIBT-resistantG2orJ3mutantsretainpartialG2orJ3activity,unlikeIBT-dependentG2orJ3mutants.1.SincetheirtranscriptlengthisinuencedbyIBTinvivo,theirIBT-resistanceisprobablynotattributableadeciencyinIBTbinding.2.BecausetheygrowinthepresenceofIBT,theymustmaketranscriptsthatareshorterthantranscriptsproducedbywild-typevirusinthepresenceofIBT,sincewild-typetranscriptsaresolongastoactivatecellular

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antiviralresponses.3.BecausetheygrowintheabsenceofIBT,theymustmaketranscriptsthatarelongerthantranscriptsproducedbyIBT-dependentmutantsintheabsenceofIBT,sinceIBT-dependentmutanttranscriptsaretooshorttoencodethelargelateproteins.AninterestingquestioniswhetherG2andJ3exerttheirelongation-promotingeectsviathesamemechanism.TheobservationthatIBTcansubstitutefortheactivityofeitherG2orJ3suggeststhattheymaybefunctionallyidentical.However,theysharenoobviousaminoacidsequencehomology.Furthermore,bothareessentialgenes,meaningthatneithercantaketheplaceoftheother.ItisnotknownwhetherIBTcouldsubstituteforbothG2andJ3proteinssimultaneously,andthisshouldbeaddressedbytheconstructionofaG2/J3doublemutantandtestingitsabilitytogrowbothinthepresenceandabsenceofIBT.IfsuchamutantisIBT-dependent(asseemslikelytobethecase),thiswouldprovidestrongevidencethatIBTdoesnotfunctionbystimulatingtheactivityofG2orJ3. 5.4 H5AnovelH5mutantvirusiscapableofgrowthinthepresenceofIBT.Thisobservationcanbeexplainedbyoneofatleasttwopossiblemechanisms.First,H5hasbeenpreviouslyreportedtostimulatelatetranscriptiongenerallyinvitro,thoughithasnotbeenshowntoeectelongation,perse[ 51 ].IfH5alsostimulateslatetranscriptioninvivo,anH5mutantvirusmightproducereducedlevelsoflatemRNAthatwouldresultinalower-than-wild-typeconcentrationofdsRNAinthecell.EventhoughthesemRNAswouldbeofalengththatwouldresultinanabortiveinfectioninthecaseofthewild-typevirus,theirlowerconcentrationrelativetoawild-typeinfectionwouldlikelypreventactivationofdsRNA-mediatedantiviraldefensessuchasthe2-5AandPKRpathways.Second,ifthepurposeoftheH5/G2interactionisforH5toregulatetheelongationactivityofG2,thenanH5mutantimpairedinthisactivitywouldbelesscapableofstimulatingG2

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Figure5{1:SummaryofallmappedG2Rmutants.Truncations(blue)andfulllengthorinternaldeletions(green)areshownasrectangles.Anarrowaboveeachrectangleindicatesthepositionofthemutation.Whereknown,theIBTsensitivity(S),resistance(R)ordependence(D)ofeachvirusdeterminedat31C,37C,and40Cislisted.

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andcouldthereforeexhibitaphenotyperesemblingthatofanIBT-resistantorIBT-dependentG2mutant.ThemutationsinH5R-1areonlyveaminoacidresiduesapart,andeachresultsinanon-conservativechangeofachargedtoanunchargedresidue.BothofthecloseproximityofthemutationstooneanotherandthefactthatbothresultinthelossofchargesuggestthepossibilitythatthesemutationsmaycompriseabindingsiteontheH5proteinforanotherfactor.DeterminingwhethertheH5Rmutantproteincaninteractwithwild-typeG2hasthepotentialtosignicantlyrevisethemodelofintermediateandlatetranscriptionelongationtoincludearoleforH5beyonditsearliercharacterizationasaweakstimulatoroflatetranscription[ 51 ]. 5.5 RNAPolymeraseThelargestandsecond-largestRNApolymerasesubunitsareconservedamongpoxvirusesandcellularorganisms.Wetookadvantageofthishomologybybuildingcomputermodelsthatpredictthestructureofrpo147andrpo132.TogetherwiththemappingofDL1-3,DL3-2,andDL10-7.1torpo147andthemappingofIBTr90,A24R-R1,andA24R-R2torpo132,alegitimatestructuralperspectiveofthevacciniavirusRNApolymerasehasbeguntoemerge.Thusfar,theexperimentaltranscriptionelongationdatagatheredbothinvivoandinvitroandthehomologymodelingcorroborateoneanother.BecauseDL1-3,DL3-2,andDL10-7.1eachrespondtoIBTinvivo,theirmutationsprobablydonotinterferewithIBTbindingandthussuggestadefectintranscription.SuchadefectwasobservedinaninvitroelongationassaywithDL1-3,DL3-2,andIBTr90(theothermutantshavenotyetbeenassayed).Theexperimentaldatathusconrmstheutilityofhomolgymodelingandsuggeststhatinsightsgainedfromthemodelmaybeaccurateandbiologicallyrelevant.Interestingly,theobservationthattworpo132mutants,DL1-3andDL10-7.1,sharethesamemutation(A535V)arguesthatthispositionisacriticaloneforregulatingtranscriptionelongation.Likewise,thesameargument

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canbemadefortworpo147mutants,IBTr90andA24R-R1.Thesemutantsalsoshareamutation(Y462H).Thus,IBThasprovenitselfagainasavaluabletoolfortheisolationofmutantswithtranscriptionelongationdefects. 5.6 ModelofIntermediateandLateTranscriptionGiventheaboveobservations,anumberofmodelscanbeconstructedtoexplainboththeregulationofintermediateandlatetranscriptionandthemechanismofactionofIBT.Onesuchmodelispresentedhere(Figure5-2).Accordingtothismodel,A18functionsinvivoasatranscriptionterminationfactor.Sincethereisapparentlynosequencespecicityforintermediateorlatetermination,anothermechanismmustbeinvokedtoexplain3'endformation.BecauseoftheabilityofA18toactasadsDNA-dependenthelicaseandpossiblyatranslocaseaswell,suchamechanismmightincludebindingofA18todsDNAdirectlyupstreamoftheRNApolymeraseandtransclocatinginadownstreamdirection.Whileintheprocessoftranslocating,ifA18encountersapausedpolymerase,itwouldinduceinthepolymeraseaconformationalchangeinanATP-hydrolysis-dependentstepthatwouldcausetermination,similartotheE.coliMfdprotein.FrequentpausingofthepolymerasewouldincreasetheprobabilityofA18collidingwithit,andthusterminationwouldoccurmostfrequentlyatsitesthatpromotepausing.SinceIBTresultsintheformationoflonger-than-normaltranscripts,itlikelyinhibitstranscriptiontermination.Thereareseveralpossiblewaysinwhichthiscouldoccur,includinginhibitionofA18/RNApolymeraseinteractionorinhibitionoftheputativeA18-associatedhostfactor.PerhapsthemostlikelytargetistheRNApolymeraseitself,asbindingofIBTtothepolymerasecouldinhibittheconformationalchangesrequiredforapolymerasetoenterthebacktrackedstate.Lendingsupporttothisidea,aG2/A18doublemutant[ 120 ]andaJ3/A18doublemutant[ 121 ]areeachIBT-sensitive,suggestingthatthetargetforIBTisnot

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G2,J3,orA18.Regardless,adenitiveanswertothequestionofthemoleculartargetofIBTawaitsthediscoveryandcharacterizationofanIBT-resistantmutantwhosetranscriptionisnotinuencedbyIBT.Alternatively,becauseseveralthiosemicarbazoneshavebeenshowntobindDNAinthepresenceofcationssuchascopper,perhapsIBTcompeteswithA18forbindingoftheDNAupstreamofthepolymeraseandthuspreventsitsterminatorfunction. 5.7 ConclusionsTheinterplaybetweenA18,G2,J3andtheRNApolymeraseisnotentirelyunderstood.However,thereisgeneticevidencesuggestingthattheseproteinsdonotacttodirectlymodulatetheactivityofoneanother.Rather,theylikelyregulatetheRNApolymerasedirectly.ThisargumentissupportedbythefactthatA18/G2andA18/J3doublemutantshaveaphenotypethatdiersfromasinglemutantinanyofthesegenes.Wereonegeneproducttoactupstreamofanotherinapathway,thenmutationofthegeneencodingthedownstreamproductshouldproducethesamephenotypeasmutationofbothgenes.OurstudyexpandsthelistoffactorsimplicatedinthecontroloftranscriptionelongationtoincludethelargestsubunitoftheRNApolymerase,rpo147,andtheH5protein.Determinationofthepreciseroleforthesefactorsandthosepreviouslyimplicatedinregulatingtranscriptionelongationrequiresabroadcollectionofmutantsandthedevelopmentofsensitivebiochemicalassaystowhichthemutantsshouldbesubjected.Signicantprogresscontinuestobemadebyseveralmembersofthelabonbothoftheseobjectives.

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Figure5{2:Modelforvacciniavirusintermediateandlatetranscriptionelongationandtermination.Thenucleicacidstrandsaredistinguishedbycolor,withtheDNAtemplatestrandinpurpleandthenon-templatestrandinblue.RedindicatesRNA.TheportionoftheRNAwithintheRNApolymeraseisshownwithadashedborder.Becauseelongationandterminationofintermediateandlategenesmaybemoreintricatelylinkedthanforearlygenes,theJ3,G2,andH5proteinsareshownhere.A.TheA18protein,possiblyinconjunctionwithahostfactor,terminatestranscriptionwithoutusingacis-actingsequence.B.BindingofIBT(complexedwithametalion)totheduplexDNAcompeteswithA18andthusinhibitstermination.C.Alternatively,bindingofIBTtotheRNApolymerasemayinhibittheA18/RNApolymeraseinteraction.

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StevenGainesCresawnwasbornonJuly21,1975,inNewportNews,Virginia.HeattendedJamesMadisonUniversityintheShenandoahValleyofwesternVirginia,graduatinginDecember,1996withabachelor'sdegreeinbiology.PerhapsitwasfatethatbroughthimtoGainesville,thecitynamedforhisancestor,GeneralEdmundPendletonGaines.PerhapshemovedtheretostartgraduateschoolintheInterdisciplinaryPrograminBiomedicalScienceswithKerryMargaretOwens,thewomanhewouldmarryonJune10,2000.Togethertheyhaveadaughter,AbigailLeaCresawn,bornonOctober16,2001,andarehappilyawaitingthearrivaloftheirsecondchild.UponnishinghisPh.D.inthespringof2005,Steveplanstocontinuehiscareerbyworkingasapost-doctoralassociate. 137