Molecular Mechanisms of Transcription Initiation and DNA Repair

项目概要/摘要 本申请描述了我们对人类病原体的基本分子途径的研究， 结核分枝杆菌（Mtb），包括转录调控和DNA修复的研究。感染 结核病每年导致1000多万新的结核病病例和150万人死亡，使其成为最致命的结核病。 感染世界。此外，这一健康危机继续因毒品的出现而加剧， 耐药菌株，这需要发现新的抗生素制剂。此外，我们正在深化和 拓宽了我们的生物物理学工作，阐明了真核生物转录起始的机制， 单分子实验结合酵母菌和人类的动力学模型。 转录负责在发育或适应过程中基因表达模式的变化。 环境条件RNA聚合酶（RNAP）在特定时间向特定基因的募集， 在转录起始期间由通用和基因特异性转录因子组执行。我们 研究结核分枝杆菌（Mycobacterium tuberculosis，CardD）的基本的、不依赖于操作者的全局转录因子， 和RbpA。这些因素通过调节异构化进出开放复合物的速率而起作用 与直觉相反，它似乎能够作为激活剂或抑制剂起作用， 直接识别DNA序列。我们将回答该领域中有关序列的关键问题， σ因子（即，应激反应）依赖这些因素，以及他们的作用后启动阶段 转录。 我们还在研究结核分枝杆菌中转录和DNA修复之间的联系。分枝杆菌缺乏典型的 保守的错配修复途径（MMR），并拥有在E.杆菌另外我们有 最近发现了一种新的激活Mtb核苷酸切除修复酶（NER）的氧化开关， UvrD1.我们目前正在研究这种开关的生物物理性质，替代激活途径， UvrD 1在转录偶联NER过程中与RNAP相互作用的能力。特别感兴趣，并提供 我们的研究之间的联系，是共同的RNAP结合位点使用的两个卡和UvrD 1。 最后，我们将继续研究前引发复合物的动力学中间体 (PIC)依赖性转录起始具体来说，我们正在确定DNA泡沫的机制， 在酵母和人类的初始转录过程中扩增。我们的单分子磁镊 实验将提供PIC功能机制的高分辨率视图。我们也在跟进 我们最近发现酵母和人TFIIH（一般转录）活性之间的差异， 启动子解旋所需的因子），这可能是这些基因中转录起始位点的不同用途的基础。 有机体由于PIC功能是基因表达的基础，我们独特的方法将提供重要的进展 在人类生物学研究中。

PROJECT SUMMARY/ABSTRACT This application describes our research into essential molecular pathways of the human pathogen, Mycobacterium tuberculosis (Mtb), including studies of transcription regulation and DNA repair. Infection with Mtb results in over 10 million new cases of tuberculosis and 1.5 million deaths annually, making it the deadliest infection in the world. In addition, this health crisis continues to be exacerbated by the emergence of drug- resistant strains, which demands the discovery of new antibiotic agents. In addition, we are deepening and broadening our biophysical work elucidating mechanisms of eukaryotic transcription initiation via both ensemble and single-molecule experiments coupled with kinetic modeling of the process in both yeast and humans. Transcription is responsible for changes in gene expression patterns during development or in adaptation to environmental conditions. The recruitment of RNA polymerase (RNAP) to particular genes at particular times is performed by sets of general and gene-specific transcription factors during transcription initiation. We are studying the essential, operator-independent, global transcription factors of Mycobacterium tuberculosis, CarD and RbpA. These factors act by modulating the rates of isomerization into and out of the open complex intermediate in initiation and, contrary to intuition, appear able to act as either activators or repressors without recognizing DNA sequence directly. We will answer critical questions in the field regarding the sequence- and sigma-factor (i.e., stress-response) dependence of these factors as well as their roles in post-initiation phases of transcription. We are also studying links between the transcription and DNA repair in Mtb. Mycobacteria lack classically conserved mismatch repair pathways (MMR) and possess repair factors not seen in E. coli. In addition, we have recently uncovered a novel oxidative switch that activates the Mtb nucleotide excision repair enzyme (NER), UvrD1. We are currently investigating the biophysical nature of this switch, alternative activation pathways, and the ability of UvrD1 to interact with RNAP during transcription-coupled NER. Of particular interest, and providing a link between our studies, is the shared RNAP-binding site used by both CarD and UvrD1. Lastly, we are continuing our investigations of the kinetic intermediates underlying pre-initiation-complex (PIC) dependent transcription initiation. Specifically, we are determining the mechanism of DNA bubble expansion during initial transcription in both yeast and humans. Our single-molecule magnetic-tweezers experiments will provide high-resolution views of the mechanism of PIC function. We are also following up on our recent discoveries of differences between the activities of yeast and human TFIIH (the general transcription factor required for promoter unwinding) that may underly the distinct usage of transcription-start sites in these organisms. As PIC function underlies gene expression, our unique approaches will provide important advances in the study of human biology.