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

基本信息

批准号：
10313703
负责人：
Robert A Battaglia
金额：
$ 6.6万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
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/10313703
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 Gap Junctions 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

项目摘要

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.

项目概要/摘要在所有生命中，基因表达取决于 RNA 忠实且及时地转录信使 RNA 聚合酶。 RNA 聚合酶 (RNAP) 延伸的分子细节已确定来自几个物种，但尚不清楚这些例子是否能代表整体。最近，RNAP 从模型中发现，革兰氏阳性细菌枯草芽孢杆菌与翻译和旅行无关其 RNAP 的速度比革兰氏阴性菌大肠杆菌的 RNAP 快得多。这个观察结果是补充了这些物种之间已知的转录调控差异。我假设由于许多基因调控机制作用于 RNAP 的延伸，因此延伸率的变化会影响 RNAP 的延伸。影响这种调节的发生方式。我的建议的目标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 RNAP 相关因子在共转录基因调控。因为暂停是体内 RNAP 延伸率的主要决定因素，我提案的目标 1 我将使用新生 RNA 确定枯草芽孢杆菌和大肠杆菌之间暂停的差异测序。在目标 2 中，我将检查影响枯草芽孢杆菌和大肠杆菌 RNAP 延伸的内在因素和反式因素使用单分子显微镜进行体外速率。在目标 3 中，我将建立枯草芽孢杆菌 RNAP- 的功能与遗传相互作用筛选相关的因素。这项工作将阐明枯草芽孢杆菌的特性转录机制，这可能是其他厚壁菌门细菌所共有的，包括病原体金黄色葡萄球菌。金黄色葡萄球菌和艰难梭菌。由于 RNAP 是既定的抗生素靶标，因此了解 RNAP 的差异跨细菌的功能可以为新型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资助者 Gene-Wei Li 博士和 Dr. Gene-Wei Li 的知识和经验。 Jeff Gelles，促进我的研究和职业目标。李博士拥有深厚的定量生物学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。盖勒斯在单分子生物物理学方面拥有多年的经验，如果我开发自己的单分子生物物理学，这将是一笔重要的财富该领域的技能。盖勒斯博士还指导了几名获得独立学位的博士后学员研究职位。我的赞助商的支持以及研究基础设施和职业生涯麻省理工学院的机会将有助于完成以下研究目标这个提议以及我作为一名独立科学家的发展。