Molecular Mechanisms of Transcription Initiation and DNA Repair

项目摘要/摘要 这项申请描述了我们对人类病原体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。感染了 结核病每年导致1000多万新结核病病例和150万人死亡，使其成为死亡人数最多的 世界上最大的传染病。此外，毒品的出现继续加剧了这场健康危机。 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 拓宽我们的生物物理工作，阐明真核转录启动的机制 以及单分子实验，结合酵母和人类的这一过程的动力学模型。 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)在特定时间对特定基因的募集是 在转录启动过程中由一组通用的和基因特异的转录因子执行。我们是 研究结核分枝杆菌基本的、非操作者依赖的全局转录因子 和RbpA。这些因素通过调节进入和离开开放复合体的异构化速率起作用 在启动中居中，与直觉相反，似乎能够在没有直觉的情况下充当激活者或抑制者 直接识别DNA序列。我们将回答该领域有关序列的关键问题--以及 这些因子的Sigma因子(即应激反应)依赖性及其在启动后阶段中的作用 抄写。 我们还在研究结核分枝杆菌的转录和DNA修复之间的联系。分枝杆菌缺乏经典 保守的错配修复途径(MMR)，并具有在大肠杆菌中未见的修复因子。此外，我们还有 最近发现了一种新的氧化开关，可以激活线粒体核苷酸切除修复酶(NER)， 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，以及 在转录偶联NER过程中，UvrD1与RNAP相互作用的能力。特别感兴趣，并提供 我们研究之间的一个联系，是CARD和UvrD1使用的共享RNAP结合位点。 最后，我们正在继续研究引发前复合体背后的动力学中间体。 (图)依赖转录起始。具体地说，我们正在确定DNA泡沫化的机制 酵母和人类在初始转录过程中的扩增。我们的单分子磁镊子 实验将提供对PIC作用机制的高分辨率观察。我们也在跟进 我们最近发现的酵母和人类TFIIH(一般转录)活性的差异 启动子解离所需的因子)，这可能低于转录起始点在这些 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 在人类生物学的研究中。

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.