Mechnanism of transcript elongation control by RfaH

超出所提供的空间。 许多基因的表达受到RNA的能力的限制，RNA完全聚合到一个核苷酸序列，在一个主要的调节步骤消失后，在起始位置出现一个延伸。已经描述了几种辅助旋转插入子，它们允许RNA聚合酶克服这种限制，并成为"延伸-特异性的"。在本研究中，细菌蛋白RnfaH是几种伸长和受精卵的调节因子，它能促进远端基因的表达。我们已经证明，RfaH与它的募集序列ops结合，ops暴露在RNA聚合酶表面，在伸长过程中暂停在opa位点上。它的募集后，RnfaH通过提高伸长速率和抑制暂停来刺激op位点的转录。RfaH的抗终止作用机制，除了与其他抗终止剂如_、N和LQ的作用机制不同外，其他抗终止剂如_、N和LQ对延长和终止都有深刻的影响，RfaH的募集方式和对延长的影响都是独特的，因此，RfaH的抗终止作用机制是不明确的。对RfaH机制的认识将有助于对细菌和真核生物中转录延长调控的一般理解，其中RfaH同源物是延伸控制和定位于活性转录位点的暗示。 在本提案中，我们将结合生物化学、遗传学和生物物理学方法来解决RfaH作用的几个方面。本项目的第一个目标是阐明RfaH影响其募集位点下游数千个核苷酸的选择性排列的血液分子机制。需要回答的中心机制问题是RfaH是否与延长的RNA聚合酶一起传播，或者它是否会引起RNA聚合酶的构象变化，从而导致RNA聚合酶的构象变化。随后RfaH从复合物中解离。这个项目的第二个目标是通过发现RfaH是否影响所有的转录位点或特定的调节系统来确定流动的机制，第三个目标是绘制RfaH和转录延长复合体之间的连接，从而将RfaH机制置于其结构中。 RfaH控制着这些分泌分子、细胞壁成分、致病因子、刺激因子和传播性大肠杆菌活动所需蛋白质的表达，因此，提出的研究将对细菌学和进化的几个领域产生积极影响，如细胞质外成分的合成、细菌感染、侧链素转移和病原体的出现。性能现场==

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SiTE ========================================Section End===========================================