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

项目摘要/摘要 在整个生命过程中，基因的表达依赖于rna对信使rna的正确和及时的转录。 聚合酶。已经确定了酶的RNA聚合酶(RNAP)延伸的分子细节 来自少数物种，但尚不清楚这些例子是否代表整个物种。最近，RNAP 从模型革兰氏阳性细菌中发现枯草芽孢杆菌在翻译和运输过程中是解偶联的 比来自革兰氏阴性菌的RNAP的速度要快得多。这一观察结果是 这些物种之间在转录调控方面的已知差异是相辅相成的。我假设 因为许多基因调节机制都作用于延长RNAP，所以延伸率的变化将 影响这一规定的实施方式。我的建议的目标是确定延伸率的原因 枯草杆菌与大肠杆菌的差异及枯草杆菌RNAP相关因子在细菌中的作用 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，在 我的提案的目标1我将使用新生RNA来确定枯草杆菌和大肠杆菌之间停顿的差异 测序。在目标2中，我将研究影响枯草杆菌和大肠杆菌RNAP延伸的内在和反式因素 用单分子显微镜进行体外培养。在目标3中，我将建立枯草杆菌RNAP的功能- 与遗传交互作用屏幕相关的因素。这项工作将阐明枯草杆菌的特性。 转录机制，可能与包括病原体S. 金黄色葡萄球菌和艰难梭菌。因为RNAP是一个既定的抗生素靶点，所以了解RNAP中的差异 跨细菌的功能可能会为新的物种特异性抗生素的开发提供信息。 我的奖学金培训计划结合了我的赞助人吉恩-魏丽博士和吉恩-魏丽博士的知识和经验。 杰夫·盖尔斯，以促进我的研究和职业目标。李博士在数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。Dr。 盖尔斯在单分子生物物理学方面有多年的经验，如果我开发自己的经验，这将是一项主要的财富 这方面的技能。盖尔斯博士还指导了几名获得独立博士学位的博士后实习生 研究职位。我的赞助商的支持与研究基础设施和事业的结合 麻省理工学院的机会将促进在#年完成研究目标 这项提议和我作为一名独立科学家的发展。

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.