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: 8695415
• 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 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8695415
• 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; public health relevance; 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。目前结核病（TB）治疗的不足之处，需要发现新的药物来治疗结核分枝杆菌感染。在以前的工作中，我们已经确定了CardD作为转录调节因子，这是结核病发病所必需的，有助于利福平耐药性，调节核糖体RNA（rRNA）水平，并不存在于真核生物中。因此，CardD是一个有吸引力的药物靶标，但需要了解CardD功能的分子细节以开发CardD活性的特异性抑制剂。我们假设，由于CardD是调节转录所必需的，那么它的结构域在转录过程中执行特定的功能，并且它们的活性可以被抑制以损害这些过程。我们将利用一种创新的单分子方法来真实的监测分枝杆菌RNA聚合酶（RNAP）从分枝杆菌rRNA启动子的转录，并确定CardD如何调节转录的每个单独阶段。具体而言，以下目标将在分子，生物化学和生物物理水平上解决CardD的机制，以深入了解Mtb的发病机制，并扩大原核转录的范例。 目标1.阐明CardD在rRNA启动子上的作用机制。我们将使用单分子技术来定量确定CardD对转录不同阶段的影响，并了解CardD如何影响转录动力学。 目标2.确定CardD对RNAP的利福平敏感性的影响。我们将测量在利福平存在下，CardD对转录起始和失败转录的详细动力学的影响。 目标3.研究转录调控过程中CardD大分子相互作用的作用。使用点突变在CardD，我们将确定如何破坏CardD，RNAP和启动子之间的大分子相互作用影响CardD的转录和利福平耐药性的调节。这项工作的结果将是一个详细的机制，CardD活动，这将提供答案的基本问题，在分枝杆菌转录调控。我们的研究将深入了解CardD的基本活性，然后可能在新的化疗策略中靶向治疗TB。值得注意的是，CardD在许多其他细菌中是保守的，这表明我们的发现将适用于不同的细菌病原体。因此，拟议的研究将推进美国国立卫生研究院的使命，以获得基础知识，减少传染病对人类健康的负担。