Structure and Function of RNA Polymerase in E.coli

描述（申请人提供）：RNA聚合酶（RNA polymerase， RNAP）是基因表达的中心酶，是基因调控的靶标。本研究的长期目标是了解RNAP作为分子机器的功能，并开发干预基因表达的方法。我们打算(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合成的调控中起作用；(2)描述了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的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。其中一些化合物也可作为抗菌开发的先导化合物。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，这些中间体对每种特定的催化活性都是最佳的。活性中心突变和测试抑制剂对RNA合成和降解的影响将被表征。功能中间体的复合物将使用我们之前开发的方法库进行研究，包括化学核酸-蛋白质交联，Fe2+介导的亲和足迹，基因工程突变和鉴别生化分析。此外，将开发新的基于发光的方法来跟踪伴随RNA聚合酶核苷酸添加周期的动态转变。结果将在高分辨率的背景下解释x射线结构的RNA聚合酶功能复合物使用分子模型。该项目产生的数据有望为RNA合成的基本催化机制及其调控提供新的见解。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靶点。公共卫生相关性：RNA聚合酶的反应机制将使用多种生化和生物物理方法进行详细表征，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的RNA聚合酶靶向抗菌剂的先导化合物。

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.