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（由申请人提供）：作为基因表达的中心酶，RNA聚合酶（RNAP）是基因调节剂的靶标。这项研究的长期目标是了解RNAP如何作为分子机器的功能，并开发用于介入基因表达的方法。我们打算（1）详细描述活性中心的特定氨基酸残基和结构特征，以及在RNA合成和降解的催化反应以及RNA合成的调节中，在活性中心功能附近如何（2）描述RNA聚合酶通过跟踪底物进入活性中心的途径选择三磷酸底物的机制；（3）表征先前通过筛选小型化合物的文库获得的新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。这些化合物中的一些也可能是抗菌发育的铅化合物。 RNA合成和降解的反应将在特定设计的RNA聚合酶功能中间体中进行研究，这些中间体最适合每种特定的催化活性。主动中心突变和测试抑制剂对RNA合成和降解的影响。功能中间体的复合物将使用我们先前开发的方法进行研究，其中包括化学核酸 - 蛋白质交联，Fe2+介导的亲和力足迹，基因工程的突变和歧视性生化测定。此外，将开发出新的基于发光的方法，以遵循伴随RNA聚合酶核苷酸添加周期的动态跃迁。结果将在使用分子建模的RNA聚合酶功能复合物的高分辨率X射线结构的背景下进行解释。该项目产生的数据有望提供有关RNA合成及其调节的基本催化机制的新见解。此外，这项研究中获得的知识将有助于对RNA聚合酶的新抑制剂的合理设计，这是抗菌化学疗法的被证明的靶标。公共卫生相关性：将使用多种生化和生物物理方法进行详细表征RNA聚合酶的反应机制，包括化学交联，亲和力足迹，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。这些抑制剂中的某些可以用作开发新的RNA聚合酶靶向抗菌剂的铅化合物。