Determinants of elongation rate differences between B. subtilis and E. coli RNA polymerases

项目总结/摘要 在所有的生命中，基因的表达依赖于信使RNA的忠实和及时的转录。 聚合酶。RNA聚合酶（RNAP）延伸的分子细节已被确定为酶 但目前还不清楚这些例子是否代表了整个物种。近日，RNAP 从模式革兰氏阳性菌枯草芽孢杆菌被发现是解偶联的翻译和旅行 以比RNAP更快的速度从革兰氏阴性对应物大肠杆菌中分离。该观察结果 这些物种之间的转录调控的已知差异补充。我假设 由于许多基因调控机制作用于RNAP的延伸，延伸速率的变化将 影响这一规定的实施。我的建议的目标是确定延伸率的原因 B之间的分歧。subtilis和E.大肠杆菌中表达，并探讨B.枯草杆菌RNAP相关因子 共转录基因调控。因为停顿是体内RNAP延伸率的主要决定因素， 目标1我的建议，我将确定之间的差异暂停B。subtilis和E.使用新生RNA的大肠杆菌 测序在目标2中，我将考察影响B的内在因素和反式因素。subtilis和E.大肠杆菌RNAP延伸 率在体外使用单分子显微镜。在目标3中，我将建立B的函数。枯草杆菌RNAP- 相关因素与遗传相互作用的筛选。这项工作将阐明B的特质。枯草 转录机制，这可能是由其他厚壁菌门细菌，包括病原体S。 aureus和C.很难由于RNAP是一个既定的抗生素靶点，了解RNAP的差异 跨细菌的功能可以为开发新的物种特异性抗生素提供信息。 我的奖学金培训计划结合了我的赞助人，李金伟博士和李博士的知识和经验。 Jeff Gelles，以促进我的研究和职业目标。李博士在定量生物学方面有很强的背景 并将为我提供测序实验设计和计算数据分析方面的培训。博士 Gelles在单分子生物物理学方面有多年的经验，如果我自己开发，这将是一个重要的资产 这方面的技能。Gelles博士还指导了几名博士后学员，他们获得了独立的 研究职位。我的赞助商的支持与研究基础设施和职业生涯相结合 在马萨诸塞州理工学院的机会将促进研究目标的完成， 这个建议和我作为一个独立科学家的发展。

PROJECT SUMMARY/ABSTRACT Across all life, gene expression depends upon the faithful and timely transcription of messenger RNAs by RNA polymerase. The molecular details of RNA polymerase (RNAP) elongation have been determined for enzymes from a few species, but it is unclear whether these examples are representative of the whole. Recently, RNAP from the model gram-positive bacterium Bacillus subtilis was found to be uncoupled from translation and travel at a much faster rate than RNAP from its gram-negative counterpart, Escherichia coli. This observation is complemented by known differences in transcriptional regulation between these species. I hypothesize that because many mechanisms of gene regulation act upon elongating RNAP, changes in elongation rate will affect how this regulation occurs. The goal of my proposal is to determine the causes of elongation rate divergence between B. subtilis and E. coli and to examine the roles of B. subtilis RNAP-associated factors in co-transcriptional gene regulation. Because pausing is a major determinant of RNAP elongation rate in vivo, in Aim 1 of my proposal I will determine differences in pausing between B. subtilis and E. coli using nascent RNA sequencing. In Aim 2, I will examine intrinsic and trans factors affecting B. subtilis and E. coli RNAP elongation rate in vitro using single-molecule microscopy. In Aim 3, I will establish functions of B. subtilis RNAP- associated factors with a genetic interaction screen. This work will illuminate the idiosyncrasies of B. subtilis transcriptional machinery, which are likely shared by other Firmicutes bacteria including the pathogens S. aureus and C. difficile. Because RNAP is an established antibiotic target, understanding differences in RNAP function across bacteria could inform the development of novel species-specific antibiotics. My fellowship training plan combines the knowledge and experience of my sponsors, Dr. Gene-Wei Li and Dr. Jeff Gelles, to facilitate my research and career goals. Dr. Li has a strong background in quantitative biology and will provide me training in the design of sequencing experiments and computational data analysis. Dr. Gelles has years of experience in single-molecule biophysics that will be a major asset has I develop my own skills in this area. Dr. Gelles has also mentored several post-doctoral trainees that have obtained independent research positions. The support of my sponsors in combination with the research infrastructure and career opportunities at the Massachusetts Institute of Technology will facilitate the completion of the research aims in this proposal and my development as an independent scientist.