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

枯草芽孢杆菌和大肠杆菌 RNA 聚合酶之间延伸率差异的决定因素

• 批准号: 10453438
• 负责人: Robert A Battaglia
• 金额: $ 6.76万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10453438
• 关键词: Affect; Antibiotics; Area; Bacillus subtilis; Bacteria; Binding; Bioinformatics; Biological; Biology; Biophysics; Characteristics; Clostridium difficile; Complement; Coupling; DNA-Directed RNA Polymerase; Data Analyses; Development; Enzymes; Escherichia coli; Fellowship; Fibrinogen; Firmicutes; Frequencies; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Gram-Positive Bacteria; Growth; Guanosine Tetraphosphate; In Vitro; Institutes; Knock-out; Knowledge; Life; Massachusetts; Measures; Mediating; Mentors; Messenger RNA; Microscopy; Modeling; Molecular; Mutagenesis; Nucleotides; Organism; Phenotype; Positioning Attribute; Postdoctoral Fellow; Process; RNA Polymerase I; Regulation; Research; Research Infrastructure; Resolution; Role; Scientist; Site; Speed; Staphylococcus aureus; Sum; Technology; Testing; Time; Training; Transcriptional Regulation; Translations; Travel; Work; career; design; environmental change; experience; experimental study; genetic approach; in vivo; lens; mutant; novel; pathogen; pathogenic bacteria; rate of change; single molecule; skills; transcriptome sequencing; transposon sequencing

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.

项目摘要/摘要 在所有生命中，基因表达取决于RNA对信使RNA的忠实和及时转录 聚合酶。 RNA聚合酶（RNAP）伸长的分子细节已确定用于酶 从一些物种中，但尚不清楚这些示例是否代表整体。最近，RNAP 从模型中，革兰氏阳性细菌枯草细菌被发现与翻译和旅行无偶联 比革兰氏阴性属性大肠杆菌的RNAP的速度要比RNAP快得多。这个观察是 这些物种之间的转录调节中已知差异的补充。我假设这一点 由于基因调节的许多机制作用于拉长RNAP，因此伸长率的变化将会 影响该调节的发生方式。我的建议的目的是确定伸长率的原因 枯草芽孢杆菌和大肠杆菌之间的差异，并检查枯草芽孢杆菌相关因子在 共转录基因调控。因为暂停是体内RNAP伸长率的主要决定因素，所以 我的建议的目标1我将使用新生的RNA确定枯草芽孢杆菌和大肠杆菌之间暂停的差异 测序。在AIM 2中，我将检查影响枯草芽孢杆菌和大肠杆菌伸长的固有和反式因素 使用单分子显微镜在体外速率。在AIM 3中，我将建立枯草芽孢杆菌的功能 与遗传相互作用筛选的相关因素。这项工作将阐明枯草芽孢杆菌的特质 转录机械可能由其他富公司分享，包括病原体。 金黄色葡萄球菌和艰难梭菌。因为RNAP是已建立的抗生素靶标，所以了解RNAP的差异 跨细菌的功能可以告知新型物种特异性抗生素的发展。 我的奖学金培训计划结合了我的赞助商Gene-Wei Li博士和博士的知识和经验。 杰夫·盖尔斯（Jeff Gelles），以促进我的研究和职业目标。 Li博士在定量生物学方面具有很强的背景 并将为我提供测序实验和计算数据分析的设计培训。博士 Gelles在单分子生物物理学方面具有多年的经验，这将是主要资产 该领域的技能。 Gelles博士还指导了几位独立的博士后学员 研究职位。我的赞助商的支持与研究基础设施和职业相结合 马萨诸塞州理工学院的机会将促进研究的目的 这一提议和我作为独立科学家的发展。