Structure and Function of RNA Polymerase in E.coli

大肠杆菌 RNA 聚合酶的结构和功能

基本信息

批准号：
7672374
负责人：
ARKADY MUSTAEV
金额：
$ 35.1万
依托单位：
UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-03-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7672374
关键词：
Acceleration Address Affect Affinity Amino Acids Aspartate Bacillus (bacterium)Bacillus cereus Biochemical Biological Assay Catalytic Domain Chemicals Collection Complex DNA-Directed RNA Polymerase Data Development Engineering Enzyme Gene Escherichia coli Fluorescence Gene Expression Gene Mutation Gene Targeting Genetic Transcription Goals Growth Hydroxyl Radical Individual Knowledge Lanthanoid Series Elements Lead Libraries Maps Mediating Metals Methods Modeling Molecular Conformation Molecular Machines Molecular Models Multi-Drug Resistance Mutation Mycobacterium tuberculosis Nucleic Acids Nucleotides Pathway interactions Proteins Pseudomonas aeruginosa RNA RNA Polymerase Inhibitor RNA chemical synthesis Reaction Regulation Regulator Genes Research Resolution Roentgen Rays Role Route Screening procedure Site Staphylococcus aureus Structure Testing Transcription Elongation Transcription Initiation Triad Acrylic Resin United States National Institutes of Health Universities Wisconsin Work antimicrobial base chemotherapy conformational conversion crosslink design drug development flexibility high throughput screening inhibitor/antagonist insight luminescence luminescence resonance energy transfer molecular modeling novel pathogenic bacteria phosphodiester protein crosslink public health relevance small molecule tool tripolyphosphate

项目摘要

DESCRIPTION (provided by applicant): RNA polymerase (RNAP), as the central enzyme of gene expression, is the target of gene regulators. The long-term objectives of this research are to understand how RNAP functions as a molecular machine and t develop methods for intervening in gene expression. We intend to (1) describe in detail how specific amino acid residues and structural features of the active center and regions proximal to the active center function in the catalytic reactions of RNA synthesis and degradation as well as in the regulation of RNA synthesis; (2) describe the mechanism by which RNA polymerase selects nucleotide triphosphate substrates by tracking the route of substrate into the active center; and (3) characterize new RNA polymerase inhibitors previously obtained by screening libraries of small compounds and evaluate those inhibitors as potential tools for investigating of the RNA polymerase catalytic mechanism. Some of these compounds may also serve as lead compounds for antimicrobial development. The reactions of RNA synthesis and degradation will be studied in specifically designed RNA polymerase functional intermediates that are optimal for each particular catalytic activity. The effects of active-center mutations and tested inhibitors on RNA synthesis and degradation will be characterized. Complexes of functional intermediates will be studied using the arsenal of approaches we developed previously, which include chemical nucleic acid-protein crosslinking, Fe2+mediated affinity footprinting, genetically engineered mutations, and discriminative biochemical assays. Moreover, new luminescence-based approaches will be developed to follow dynamic transitions that accompany the nucleotide addition cycle of RNA polymerase. The results will be interpreted in the context of high resolution X-ray structures of RNA polymerase functional complexes using molecular modeling. The data generated by this project are expected to provide new insights into the basic catalytic mechanism of RNA synthesis and its regulation. In addition, the knowledge gained in this study will assist rational design of new inhibitors of RNA polymerase, which is a proven target for antimicrobial chemotherapy. PUBLIC HEALTH RELEVANCE: The reaction mechanism of RNA polymerase will be characterized in detail using a variety of biochemical and biophysical methods that include chemical cross-linking, affinity footprinting, genetic mutations, and new luminescence approaches. This work will be extended by characterizing specific small-molecule inhibitors previously obtained. Some of these inhibitors may serve as lead compounds for developing new RNA polymerase-targeted antimicrobials.

描述（申请人提供）：RNA聚合酶（RNAP）作为基因表达的中心酶，是基因调控的靶点。这项研究的长期目标是了解 RNAP 作为分子机器如何发挥作用，并开发干预基因表达的方法。我们的目的是：（1）详细描述活性中心和活性中心附近区域的特定氨基酸残基和结构特征如何在RNA合成和降解的催化反应以及RNA合成的调控中发挥作用； (2)描述RNA聚合酶通过追踪底物进入活性中心的路径来选择三磷酸核苷酸底物的机制； (3) 表征先前通过筛选小化合物文库获得的新型 RNA 聚合酶抑制剂，并评估这些抑制剂作为研究 RNA 聚合酶催化机制的潜在工具。其中一些化合物还可以作为抗菌药物开发的先导化合物。 RNA 合成和降解反应将在专门设计的 RNA 聚合酶功能中间体中进行研究，这些中间体对于每种特定的催化活性都是最佳的。活性中心突变和测试的抑制剂对 RNA 合成和降解的影响将得到表征。功能中间体复合物将使用我们之前开发的方法进行研究，其中包括化学核酸-蛋白质交联、Fe2+介导的亲和足迹、基因工程突变和判别性生化测定。此外，将开发新的基于发光的方法来跟踪伴随RNA聚合酶的核苷酸添加循环的动态转变。将使用分子模型在 RNA 聚合酶功能复合物的高分辨率 X 射线结构的背景下解释结果。该项目产生的数据有望为RNA合成及其调控的基本催化机制提供新的见解。此外，本研究中获得的知识将有助于合理设计新的 RNA 聚合酶抑制剂，RNA 聚合酶是抗菌化疗的一个经过验证的靶点。公共健康相关性：将使用各种生物化学和生物物理方法详细描述 RNA 聚合酶的反应机制，包括化学交联、亲和足迹、基因突变和新的发光方法。这项工作将通过表征先前获得的特定小分子抑制剂来扩展。其中一些抑制剂可以作为开发新型 RNA 聚合酶靶向抗菌药物的先导化合物。