Molecular Mechanisms of Transcription Initiation and DNA Repair

项目概要/摘要 该应用程序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 Mtb 每年导致超过 1000 万新发结核病例和 150 万人死亡，使其成为最致命的结核病 世界上的感染。此外，这一健康危机因毒品的出现而继续加剧。 耐药菌株，这需要发现新的抗生素药物。此外，我们正在深化和 扩大我们的生物物理工作，通过两个整体阐明真核转录起始机制 以及单分子实验以及酵母和人类过程的动力学模型。 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) 在特定时间向特定基因的募集是 在转录起始过程中由一组通用和基因特异性转录因子执行。我们是 研究结核分枝杆菌必需的、独立于操作者的全局转录因子 CarD 和 RbpA。这些因素通过调节异构化进出开放络合物的速率来起作用 中间启动，与直觉相反，似乎能够充当激活剂或阻遏剂，而无需 直接识别DNA序列。我们将回答该领域有关序列和 这些因素的西格玛因素（即应激反应）依赖性及其在启动后阶段的作用 的转录。 我们还在研究 Mtb 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) 并拥有大肠杆菌中未见的修复因子。此外，我们还有 最近发现了一种新型氧化开关，可以激活 Mtb 核苷酸切除修复酶（NER）， 紫外线D1。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 UvrD1 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，我们正在继续研究预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 气泡的机制 在酵母和人类的初始转录过程中进行扩增。我们的单分子磁力镊子 实验将为 PIC 功能机制提供高分辨率的视图。我们也在跟进 我们最近发现酵母和人类 TFIIH（一般转录 启动子解旋所需的因子）可能是这些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，我们独特的方法将带来重要进展 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.