Regulation of transcription elongation

转录延伸的调控

• 批准号: 10459526
• 负责人: PAUL L BABITZKE
• 金额: $ 31.93万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10459526
• 关键词: 5' Untranslated Regions; Active Sites; Affect; Bacillus subtilis; Bacteria; Base Pairing; Binding; Biological Assay; Biological Models; Cell physiology; Collaborations; Computer Analysis; Coupling; Cryoelectron Microscopy; DNA; DNA-Directed RNA Polymerase; Data; Digestion; Elements; Escherichia coli; Event; Exhibits; Factor Analysis; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Health; Human; In Vitro; Mediating; Methods; Modeling; Open Reading Frames; Organism; Pancreatic ribonuclease; Play; Positioning Attribute; Prevalence; Process; Proteins; RNA; RNA Folding; RNA analysis; Regulation; Role; Side; Sigma Factor; Signal Transduction; Site; Testing; Time; Transcript; Transcription Elongation; Transcriptional Elongation Factors; Transcriptional Regulation; Translations; Visualization; Work; flexibility; follow-up; genome-wide; human disease; improved; in vivo; insight; interest; promoter; response; rho; stem; termination factor; transcription termination; transcriptome sequencing; transcriptomics

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.

项目摘要/摘要 RNA聚合酶（RNAP）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是两个能够刺激的一般转录伸长因子 暂停和终止细菌。暂停可以同步RNAP的位置与RNA折叠 和/或调节因子结合。使用将新生伸长的转录本测序与 RNase I消化（RNET-SEQ），确定依赖NUSG的暂停在整个整个地点发生在1,600个地点 枯草芽孢杆菌基因组。这些停顿站点中有25％在5'Utrs中，这5'UTR停顿中的几个作用 将检查在调节下游基因表达中。其他75％的停顿网站都在公开 读取框架以及其中一些暂停参与保持转录耦合的可能性 并将测试翻译。 NUSG依赖性暂停的结构基础也将使用 冷冻电子显微镜（Cryo-EM）。 NUSA和NUSG在暂停中的体内角色尚未在 大肠杆菌是因为两种蛋白质都是必不可少的。最近将探索替换NUSA和NUSG的能力 使用RNET-SEQ检查大肠杆菌中NUSA依赖性和NUSG依赖性暂停。发起人代理暂停 是一种独特的暂停机制，是由σ因子相互作用与-10启动子元素或-10样介导的 DNA中的序列。这些回溯的停顿被刺激RNA裂解的GRE因子缓解 RNAP的活动。裂解后，RNA 3'端在活动位点正确对齐，以便 伸长可以恢复。枯草芽孢杆菌近端暂停σa依赖性启动子的患病率以及 Grea在此过程中所扮演的角色将使用RNET-SEQ进行研究。 通常认为固有和依赖性依赖性终止是通过两个不同的和非重叠的 机制。固有的终结剂由RNA发夹组成，然后是富含U的道。使用3'末端映射 策略（术语seq）表明，NUSA和NUSG充当B中的一般内在终止因子。 NUSA依赖性终结剂的RNA发夹和/或不良的U诱饵，而NUSG依赖性依赖性 终结者需要依赖NUSG的暂停，以提供足够的时间来折叠具有弱A-U基座的发夹 在茎底部成对。在Rho依赖性终止中，Rho促进了转录本捕获的释放 暂停rnap。大肠杆菌NUSG参与者在某些RHO依赖性终止事件中通过用作桥梁 在RNAP和RHO之间。特别有趣的是，枯草芽孢杆菌Rho充当第三个固有终止因子，在 与公认的观点形成鲜明对比：Rho仅参与规范的Rho依赖性终止。这 依赖Rho的内在终止机制，以及NUSA或NUSG在此过程中的潜在作用，将 被探索。此外，术语seq将用于执行全面的转录组分析以确定 NUSA，NUSG和RHO在整个大肠杆菌基因组中终止的作用。然后选择的终结者将 在体外检查。