INVESTIGATING NOVEL MECHANISMS OF TRANSCRIPTION INITIATION REGULATION IN MYCOBACT

描述(申请人提供)：每年全球有180万人死于结核分枝杆菌(Mtb)。目前结核病治疗的不足要求发现治疗结核分枝杆菌感染的新药物。在以前的工作中，我们已经确定CARD是结核分枝杆菌发病所必需的转录调节因子，有助于利福平耐药，调节核糖体RNA(RRNA)水平，并且在真核细胞中不存在。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出特定的CARD活性抑制剂。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执行特定的功能，它们的活性可以被抑制以影响这些过程。我们将利用一种创新的单分子方法来实时监测分枝杆菌RNA聚合酶(RNAP)从分枝杆菌rRNA启动子中进行的转录，并确定CARD如何调控转录的每个单独阶段。具体地说，以下目的将在分子、生化和生物物理水平上探讨CARD的机制，以深入了解结核分枝杆菌的发病机制，并扩大原核转录的范式。目的1.阐明CARD对rRNA启动子的作用机制。我们将使用单分子技术来定量确定CARD在转录的不同阶段的作用，并了解CARD如何影响转录动力学。目的2.测定CARD对RNAP利福平敏感性的影响。我们将测量在利福平存在的情况下，CARD对转录启动和终止转录的详细动力学的影响。目的3.研究CARD大分子相互作用在转录调控中的作用。利用CARD中的点突变，我们将确定CARD、RNAP和启动子之间的大分子相互作用的中断如何影响CARD的转录调控和利福平耐药性。这项工作的结果将是CARD活性的详细机制，这将为分枝杆菌转录调控的基本问题提供答案。我们的研究将使我们深入了解CARD的基本活性，然后可能成为治疗结核病的新化疗策略的靶点。值得注意的是，CARD在许多其他细菌中都是保守的，这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。

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.