Structure and Function of RNA Polymerase in E.coli

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作为分子机器的功能，并开发干预基因表达的方法。我们的目的是：（1）详细描述活性中心及其邻近区域的特定氨基酸残基和结构特征在RNA合成和降解的催化反应以及RNA合成的调节中的作用;（2）描述RNA聚合酶通过跟踪底物进入活性中心的路径选择三磷酸核苷酸底物的机制;和（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，并评价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以作为抗菌剂开发的先导化合物。RNA合成和降解的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被表征。复合物的功能性中间体将使用我们以前开发的方法，其中包括化学核酸-蛋白质交联，Fe 2+介导的亲和足迹，基因工程突变，和歧视性的生化测定的武器库进行研究。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。结果将被解释的背景下，高分辨率的X-射线结构的RNA聚合酶功能复合物使用分子建模。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，在这项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，这是一个被证明的抗菌化疗的目标。公共卫生相关性：RNA聚合酶的反应机制将使用各种生物化学和生物物理方法，包括化学交联，亲和足迹，基因突变和新的发光方法进行详细描述。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。这些抑制剂中的一些可以作为开发新的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.