Regulation of transcription elongation

项目总结/摘要 RNA聚合酶（RNAP）的暂停和终止是所有基因表达的重要组成部分。 有机体NusA和NusG是两种通用的转录延伸因子，其能够刺激转录因子的转录。 在细菌中暂停和终止。暂停允许RNAP的位置与RNA折叠同步 和/或调节因子结合。使用结合新生延伸转录物测序和 通过RNase I消化（RNET-seq），确定NusG依赖性暂停发生在整个1，600个位点处。 B。枯草杆菌基因组这些暂停位点中的约25%位于5'UTR中，并且这些5'UTR暂停中的几个的作用是： 将检查在调节下游基因表达中的作用。其余75%的暂停站点处于开放状态 阅读框架和这些停顿中的一些参与维持转录偶联的可能性 翻译将受到考验。NusG依赖暂停的结构基础也将使用 冷冻电镜（Cryo-EM）。NusA和NusG在暂停中的体内作用尚未在本发明中探索。 e.因为这两种蛋白质都是必需的。最近耗尽NusA和NusG的能力将被利用， 在E. coli中，使用RNET-seq.启动子近端停顿 是一种独特的暂停机制，由σ因子与-10启动子元件或-10样启动子元件相互作用介导。 DNA中的序列这些回溯的停顿被刺激RNA切割的Gre因子所缓解 RNAP的活动。切割后，RNA 3 '端在活性位点中正确对齐，使得 可以恢复伸长。B中σ A依赖性启动子近端停顿的发生率。枯草杆菌，以及 GreA在这一过程中发挥的作用，将使用RNET-seq进行研究。 内源性和Rho依赖性终止通常被认为是通过两个不同的和非重叠的 机制等固有终止子由RNA发夹和富含U的片段组成。使用3 '末端映射 策略（Term-Seq），显示NusA和NusG在B中作为一般内在终止因子起作用。 枯草杆菌。NusA依赖性终止子具有弱的RNA发夹和/或差的U-束，而NusG依赖性终止子具有弱的RNA发夹和/或差的U-束。 终止子需要NusG依赖性停顿，以提供足够时间用于折叠具有弱A-U碱基的发夹 在茎的底部有一对。在Rho依赖性终止中，当Rho捕获到 直到暂停的RNAP。e. coliNusG作为桥梁参与了一些依赖Rho的终止事件 RNAP和Rho之间的关系特别感兴趣的是，B.枯草杆菌Rho作为第三种内在终止因子， 与Rho仅参与规范Rho依赖性终止的公认观点形成鲜明对比。的 Rho依赖性内在终止的机制，以及NusA或NusG在此过程中的潜在作用， 被探索。此外，Term-seq将用于进行全面的转录组学分析，以确定 NusA、NusG和Rho在E. coli基因组。然后，选定的终结者将 在体外进行检查。

Project Summary/Abstract RNA polymerase (RNAP) pausing and termination are important components of gene expression in all organisms. NusA and NusG are two general transcription elongation factors that are capable of stimulating pausing and termination in bacteria. Pausing allows synchronization of the position of RNAP with RNA folding and/or regulatory factor binding. Using a method that combines nascent elongating transcript sequencing with RNase I digestion (RNET-seq), it was determined that NusG-dependent pausing occurs at 1,600 sites throughout the B. subtilis genome. ~25% of these pause sites are in 5'UTRs, and the role that several of these 5'UTR pauses have in regulating downstream gene expression will be examined. The other 75% of the pause sites are in open reading frames and the possibility that some of these pauses are involved in maintaining coupling of transcription and translation will be tested. The structural basis for NusG-dependent pausing will also be investigated using cryo-electron microscopy (Cryo-EM). The in vivo roles of NusA and NusG in pausing have not been explored in E. coli because both proteins are essential. The recent ability to deplete NusA and NusG will be exploited to examine NusA-dependent and NusG-dependent pausing in E. coli using RNET-seq. Promoter proximal pausing is a distinct pausing mechanism that is mediated by σ factor interaction with -10 promoter elements or -10-like sequences in the DNA. These backtracked pauses are relieved by Gre factors that stimulate the RNA cleavage activity of RNAP. Following cleavage, the RNA 3' end becomes properly aligned in the active site such that elongation can resume. The prevalence of σA-dependent promoter proximal pausing in B. subtilis, as well as the role that GreA plays in this process, will be investigated using RNET-seq. Intrinsic and Rho-dependent termination are generally thought to occur via two distinct and non-overlapping mechanisms. Intrinsic terminators consist of an RNA hairpin followed by a U-rich tract. Using a 3' end-mapping strategy (Term-seq) it was shown that that NusA and NusG function as general intrinsic termination factors in B. subtilis. NusA-dependent terminators have weak RNA hairpins and/or poor U-tracts, whereas NusG-dependent terminators require NusG-dependent pausing to provide sufficient time for folding of hairpins with weak A-U base pairs at the bottom of the stem. In Rho-dependent termination, Rho promotes transcript release when it catches up to paused RNAP. E. coli NusG participates in some Rho-dependent termination events by serving as a bridge between RNAP and Rho. Of particular interest, B. subtilis Rho functions as a third intrinsic termination factor, in stark contrast to the accepted view that Rho only participates in canonical Rho-dependent termination. The mechanism of Rho-dependent intrinsic termination, and the potential role of NusA or NusG in this process, will be explored. In addition, Term-seq will be used to perform a comprehensive transcriptomic analysis to determine the roles of NusA, NusG and Rho on termination throughout the E. coli genome. Selected terminators will then be examined in vitro.