INVESTIGATING NOVEL MECHANISMS OF TRANSCRIPTION INITIATION REGULATION IN MYCOBACT

研究 Mycobact 中转录起始调控的新机制

• 批准号: 8881231
• 负责人: Eric A Galburt
• 金额: $ 28.88万
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8881231
• 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; 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）感染会导致全球180万人死亡。目前的结核病（TB）疗法的不足要求发现新药物以治疗MTB感染。在先前的工作中，我们将卡确定为MTB发病机理所必需的转录调节剂，有助于利福平耐药性，调节核糖体RNA（RRNA）水平，而在真核生物中不存在。因此，卡是一个有吸引力的药物靶标，但是需要了解卡功能的分子细节才能开发出卡活性的特定抑制剂。我们假设由于调节转录需要卡，因此其结构域在转录过程中执行特定功能，并且可以抑制其活性以损害这些过程。我们将利用一种创新的单分子方法来实时通过分枝杆菌RRNA启动子来监测分枝杆菌RNA聚合酶（RNAP）的转录，并确定卡如何调节每个单个转录阶段。具体而言，以下目的将在分子，生化和生物物理水平上解决卡的机制，以深入了解MTB发病机理并扩大原核生物转录的范式。 目标1。阐明卡在rRNA启动子上的作用机理。我们将使用单分子技术定量确定卡在不同转录阶段的影响，并了解卡如何影响转录动力学。 AIM 2。确定卡对RNAP的利福平灵敏度的影响。我们将测量卡片在利福平存在下转录起始和流产转录的详细动力学的影响。 目标3。调查转录调控过程中的角色O卡大分子相互作用。使用卡中的点突变，我们将确定卡，RNAP和启动子之间的大分子相互作用的破坏会影响转录和利福平耐药性的卡片调节。这项工作的结果将是卡活动的详细机制，该机制将为分枝杆菌中有关转录调节的基本问题提供答案。我们的调查将洞悉卡的基本活动，然后在治疗结核病的新化学治疗策略中可能针对这些活动。值得注意的是，卡片在许多其他细菌中都是保守的，表明我们的发现将适用于各种细菌病原体。因此，拟议的研究将促进美国国立卫生研究院的使命，以获取基本知识，以减轻传染病对人类健康的负担。