Molecular Mechanisms of Transcription Initiation and DNA Repair

转录起始和DNA修复的分子机制

• 批准号: 10581660
• 负责人: Eric A Galburt
• 金额: $ 46.85万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10581660
• 关键词: Antibiotics; Binding Sites; Biochemical Pathway; Biochemistry; Biophysics; Cessation of life; Complex; Coupled; DNA; DNA Repair; DNA Sequence; DNA-Directed RNA Polymerase; Dependence; Development; Drug resistance; Escherichia coli; Eukaryota; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Genus Mycobacterium; Health; Human; Human Biology; Infection; Intuition; Investigation; Isomerism; Kinetics; Knowledge; Link; Magnetism; Mismatch Repair; Molecular; Mycobacterium tuberculosis; Nature; Nucleotide Excision Repair; Organism; Pathway interactions; Phase; Process; Research; Resolution; Role; Sigma Factor; Transcription Initiation; Transcription Initiation Site; Transcription-Coupled Repair; Transcriptional Regulation; Tuberculosis; Work; Yeasts; biological adaptation to stress; biophysical model; experimental study; human pathogen; interest; kinetic model; novel; pathogenic bacteria; promoter; recruit; repair enzyme; repaired; resistant strain; single molecule; transcription factor; transcription factor TFIIH

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.

项目摘要/摘要 该应用描述了我们对人类病原体必需分子途径的研究， 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1000万例新的结核病和150万例死亡，使其成为最致命的 世界上的感染。此外，由于药物的出现，这种健康危机仍在加剧 抗性菌株，要求发现新的抗生素剂。此外，我们正在加深和 扩大我们通过两个合奏的真核转录开始阐明真核转录启动的机制 和单分子实验以及酵母和人类过程的动力学建模。 转录负责开发过程中基因表达模式的变化或适应 环境条件。在特定时间，RNA聚合酶（RNAP）募集到特定基因的是 在转录启动过程中由一组通用和基因特异性转录因子进行。我们是 研究结核分枝杆菌的基本，独立的全球转录因子，卡片 和RBPA。这些因素通过调节式和外部复合物的异构化速率来起作用 启动和与直觉相反的中间体似乎能够充当激活因子或阻遏物 直接识别DNA序列。我们将回答有关序列和序列的关键问题 - 这些因素及其在发射后阶段的作用的Sigma因子（即应力反应）依赖性 转录。 我们还在研究MTB中转录和DNA修复之间的联系。分枝杆菌经典缺乏 保守的不匹配修复途径（MMR），并且具有大肠杆菌中未见的修复因子。此外，我们还有 最近发现了一种新的氧化开关，该开关激活MTB核苷酸切除酶（NER），，， UVRD1。我们目前正在研究此开关的生物物理性质，替代激活途径和 在转录耦合NER期间，UVRD1与RNAP相互作用的能力。特别有趣，并提供 我们的研究之间的联系是卡和UVRD1都使用的共享RNAP结合位点。 最后，我们正在继续对原始前复合的动力学中间体进行调查 （PIC）依赖转录启动。具体而言，我们正在确定DNA气泡的机制 酵母和人类初始转录期间的扩张。我们的单分子磁性分子 实验将提供PIC功能机理的高分辨率视图。我们也在跟进 我们最近发现酵母和人类tfiih活动之间差异的发现（一般转录 启动子解放所需的因素）可能是基本的转录启动站点的不同用法 有机体。由于PIC功能是基因表达的基础，我们的独特方法将提供重要的进步 在人类生物学研究中。