Regulation of transcription elongation

项目摘要/摘要 RNA聚合酶(RNAP)的暂停和终止是ALL基因表达的重要组成部分 有机体。NusA和NusG是两种常见的转录延伸因子，能够刺激 细菌中的停顿和终止。暂停允许RNAP的位置与RNA折叠同步 和/或调节因子结合。使用一种结合了新生的延长转录测序和 RNaseI消化(rnet-seq)，已确定在整个1,600个位置发生NusG依赖的暂停 枯草杆菌基因组。这些停顿位置中约25%位于5‘UTR，其中几个停顿位置的作用 在调控下游基因表达方面将进行研究。其他75%的暂停站点是开放的 阅读框架以及其中一些停顿参与维持转录偶联的可能性 并将对翻译进行测试。NusG依赖的暂停的结构基础也将使用 低温电子显微镜(Cryo-EM)。NusA和NusG在体内对暂停的作用还没有被探索 大肠杆菌，因为这两种蛋白质都是必需的。最近消耗Nusa和NusG的能力将被利用来 用rnet-seq检测大肠杆菌中NusA依赖和NusG依赖的暂停。启动子近端停顿 是一种独特的停顿机制，由σ因子与-10启动子元件或-10类似物相互作用而介导 DNA中的序列。这些回溯的停顿被刺激RNA切割的GRE因子缓解 RNAP活性。在切割之后，RNA3‘端在活性部位中变得正确排列，从而 伸长可以恢复。枯草杆菌中依赖于σA的启动子近端停顿的发生率以及 将使用rnet-seq调查grea在这一进程中所起的作用。 固有的和依赖于Rho的终止通常被认为是通过两个截然不同且不重叠的 机械装置。内在终止子由一个RNA发夹和一个富U区组成。使用3‘末端映射 策略(Term-Seq)研究表明，NusA和NusG在B. 枯草杆菌。依赖于NusA的终止子具有弱的RNA发夹和/或较差的U-链，而依赖于NusG的终止子 终止子需要依赖NusG的暂停，以便为A-U碱基较弱的发夹的折叠提供足够的时间 对在茎的底部。在Rho依赖的终止中，Rho在捕获时促进转录释放 最多暂停RNAP。E.coliNusG作为桥梁参与一些Rho依赖的终止事件 在RNAP和Rho之间。特别值得注意的是，枯草杆菌Rho作为第三种内在终止因子，在 与公认的观点形成鲜明对比的是，Rho只参与规范的Rho依赖的终止。这个 依赖于Rho的内在终止机制，以及NusA或NusG在这一过程中的潜在作用，将 被探索。此外，将使用Term-Seq进行全面的转录分析，以确定 NusA、NusG和Rho在整个大肠杆菌基因组终止中的作用。然后，选定的终结者将 在体外进行检测。

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.