Mechnanism of transcript elongation control by RfaH

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ressionof many genes islimitedby theabilitoyf RNA p.o.ly_merasteo complete polymerizationof up to a ionnuclcotidesm,aking elongationto emerge next to initiatioans a major regulatorys_tepm gone . expression.Severalaccessory_rotointactorsthatallow RNA polymerase to overcome thisllmitatioannd become "elongation-proficienhtave been desc.n.'beTdh.e focusof thisproposal,thebactcfiatproteiRnfaH, isa regulatorof severalong virulenceand fertiliotpyerons,where itprcterentialliyncreasesme expression of distalgenes.We have demonstratedthatRfaH binds to itsrecruitmentsequence,ops,exposed on _e surfaceof theRNA polymerase paused atan opa siteduringelongation.Following itsrecruitmentR,f.aH stimulatestranscriptiodnownstream of an ops sitebyonhancing elongationrateand suppressingpausing. However, RfaH only modestly inhibittserminationT.he detailedmechanism of RfaH action,descnbcctas "antiterminationr'e,mains obscure exceptforthefactthatitisdifferent.frotmhoseof otherantiterminators such as _,Nand LQ, which have profound effectson both elongationand terminationB.oth therecruitment mode and theeffectof RfaH on elongationareunique,thus!nsightsintothe RfaH mechanism willcontribute to thegeneralunderstandingof theregulationof transcripetlongationin bacteriand alsoin eukary0tes, where RfaH homologs arcImplicatedm elongationcontrolandlocalizeto theactivelytranscribedsites. In thisproposal,we willuse a combinationof biochemical,genetic,and biophysicalapproachesto addressseveralaspectsof RfaH action.Thefirstgoal of thisprojectisto c_lucidatehemolecularmechanism by which RfaH affectselongationthousandsofnucleotidesdownstream of itsrecruitmentsite.The central mechanisticquestionto be answered iswhether RfaH travelswith the elongatingRNA polym.eraseor .itt causesa conformationalchange inthe RNA polymerase thatperslstsforthous.andsofnuclc.otldaeddison . stepsafterRfaH dissociatefsrom thecomplex. The second goal of thisprojectisto determineflow umversai isthismechanism by findin_out whetherRfaH affectstranscriptiosnimilarlayt allsitesor istargetedto a particulasretof regulatoryslgnals.The thirdgoal of thisprojectisto map inte_ctionsbetween RfaH and the transcriptioenlongationcomplex, thusplacingRt'aHmechanism in itstructuraclontext. RfaH controlstheexpressionof thesecretedmolecules,components of thecellwall,antibioticsv,irulence factors,and proteinsrequiredforthemobilizationof transmissiblpelasmids.Proposed studieswilltherefore positivelyimpact researchin severalareasof bacteriabliology and evolution,such as synthesisof extracytoplasmicomponents, bacteriavlindence,lateraglone transfera,nd emergence of pathogens.