INVESTIGATING NOVEL MECHANISMS OF TRANSCRIPTION INITIATION REGULATION IN MYCOBACTERIA

研究分枝杆菌转录起始调控的新机制

• 批准号: 9266954
• 负责人: Eric A Galburt
• 金额: $ 6.36万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-08 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9266954
• 关键词: Address; Affect; Bacillus anthracis; Bacteria; Binding; Biochemical; Biological Assay; Biological Models; Borrelia burgdorferi; C-terminal; Cessation of life; Clostridium difficile; Communicable Diseases; Complex; DNA; DNA Binding Domain; DNA-Directed RNA Polymerase; Development; Disease; Drug Targeting; Drug resistance; Eukaryota; Future; Genetic Transcription; Genus Mycobacterium; Health; Human; In Vitro; Individual; Investigation; Kinetics; Knowledge; Lead; Learning; Magnetism; Measures; Mission; Molecular; Monitor; Multi-Drug Resistance; Mutation; Mycobacterium leprae; Mycobacterium tuberculosis; N-terminal; New Agents; Outcome; Pathogenesis; Pathway interactions; Phase; Phenotype; Point Mutation; Polymerase; Population; Process; Regulation; Reporting; Research; Ribosomal RNA; Rifampicin resistance; Rifampin; Role; Staging; Techniques; Time; Transcription Initiation; Transcriptional Regulation; Tuberculosis; United States National Institutes of Health; Work; World Health Organization; in vivo; inhibitor/antagonist; innovation; insight; mycobacterial; novel; novel therapeutics; pathogen; promoter; research study; resistant strain; single molecule; tuberculosis treatment

DESCRIPTION (provided by applicant): Each year, Mycobacterium tuberculosis (Mtb) infection causes 1.8 million deaths worldwide. The inadequacies of present tuberculosis (TB) therapies demand the discovery of new agents to treat Mtb infection. In prior work, we have identified CarD as a transcriptional regulator that is necessary for Mtb pathogenesis, contributes to rifampicin resistance, regulates ribosomal RNA (rRNA) levels, and is not present in eukaryotes. CarD is thus an attractive drug target, but knowledge of the molecular details of CarD function is required to develop specific inhibitors of CarD activity. We hypothesize that since CarD is required for regulating transcription, then its structural domains perform specific functions during transcription and their activity can be inhibited to compromise these processes. We will utilize an innovative single-molecule approach to monitor transcription by mycobacterial RNA polymerase (RNAP) from mycobacterial rRNA promoters in real time and determine how CarD modulates each individual phase of transcription. Specifically, the following aims will address the mechanism of CarD at the molecular, biochemical, and biophysical levels to gain insight into Mtb pathogenesis and to expand paradigms of prokaryotic transcription. Aim 1. Elucidate the mechanism of action of CarD at rRNA promoters. We will use single molecule techniques to quantitatively determine the effect of CarD on different stages of transcription and learn how CarD affects transcription kinetics. Aim 2. Determine the effect of CarD on rifampicin sensitivity of RNAP. We will measure the effect of CarD on the detailed kinetics of transcription initiation and abortive transcription in the presence of rifampicin. Aim 3. Investigate the role o CarD macromolecular interactions during transcription regulation. Using point mutations in CarD, we will determine how disruptions in the macromolecular interactions between CarD, RNAP, and the promoter affect CarD regulation of transcription and rifampicin resistance. The outcome of this work will be a detailed mechanism of CarD activity, which will provide answers to fundamental questions regarding transcription regulation in mycobacteria. Our investigations will generate insight into the essential activity of CarD that may then be targeted in new chemotherapeutic strategies to treat TB. Notably, CarD is conserved in many other bacteria, indicating that our findings will apply to diverse bacterial pathogens. Thus, the proposed research will advance the mission of the National Institutes of Health to gain fundamental knowledge to decrease the burden of infectious disease on human health.

描述（由申请人提供）：每年，结核分枝杆菌 (Mtb) 感染导致全球 180 万人死亡。现有结核病 (TB) 疗法的不足之处需要发现新的药物来治疗 Mtb 感染。在之前的工作中，我们已经确定 CarD 是 Mtb 发病机制所必需的转录调节因子，有助于利福平耐药性，调节核糖体 RNA (rRNA) 水平，并且在真核生物中不存在。因此，CarD 是一个有吸引力的药物靶点，但需要了解 CarD 功能的分子细节才能开发 CarD 活性的特异性抑制剂。我们假设，由于 CarD 是调节转录所必需的，因此它的结构域在转录过程中发挥特定的功能，并且可以抑制它们的活性以损害这些过程。我们将利用创新的单分子方法实时监测分枝杆菌 rRNA 启动子的分枝杆菌 RNA 聚合酶 (RNAP) 的转录，并确定 CarD 如何调节每个单独的转录阶段。具体而言，以下目标将从分子、生化和生物物理水平上探讨 CarD 的机制，以深入了解 Mtb 发病机制并扩展原核转录范式。 目标 1. 阐明 CarD 对 rRNA 启动子的作用机制。我们将利用单分子技术定量测定CarD对转录不同阶段的影响，并了解CarD如何影响转录动力学。 目标 2. 确定 CarD 对 RNAP 利福平敏感性的影响。我们将测量 CarD 在利福平存在的情况下对转录起始和转录失败的详细动力学的影响。 目标 3. 研究 CarD 大分子相互作用在转录调控过程中的作用。利用 CarD 中的点突变，我们将确定 CarD、RNAP 和启动子之间大分子相互作用的破坏如何影响 CarD 对转录和利福平耐药性的调节。这项工作的成果将是 CarD 活性的详细机制，它将为有关分枝杆菌转录调控的基本问题提供答案。我们的研究将深入了解 CarD 的基本活性，然后可以将其作为治疗结核病的新化疗策略的目标。值得注意的是，CarD 在许多其他细菌中是保守的，这表明我们的研究结果将适用于多种细菌病原体。因此，拟议的研究将推进美国国立卫生研究院的使命，即获得基础知识，以减轻传染病对人类健康的负担。