Structure and Function of RNA Polymerases in E Coli

描述（申请人提供）：RNA聚合酶（RNAP）是基因表达的主要酶，也是遗传调控的靶点。这项研究的长期目标是了解细菌RNAP作为原子水平分辨率的分子机器的功能。具体而言，本研究的目的是：（1）建立一个活性中心模型，将功能分配给RNA合成和降解反应中的特定氨基酸残基、核苷酸和金属离子，并从结构上解释这些反应之间的相互关系;（2）表征调节催化功能的三元转录复合物中的构象转变;（3）理解在新生转录物的初始构建、起始因子σ的释放和启动子清除期间发生的初始转录复合物中的转换;（4）通过构建核酸适体来探索RNAP分子的可塑性，从而可以捕获RNAP的多种构象用于晶体学研究。为此，将产生一系列功能上确定的复合物，其中RNAP将（a）在转录过程的特定阶段停止;或（B）与确定的核酸支架复合，或（c）与结合的适体一起冷冻在复合物中。将使用化学核酸-蛋白质交联、RNAP基因工程突变和区分性生化测定来研究复合物。结果将解释与分子建模的帮助。在这项研究中产生的基本转录机制的理解是一个先决条件的调控现象的分子解释和发展的干预到基因表达。此外，这些研究中获得的知识将有助于合理设计和筛选新的RNAP抑制剂，RNAP已被证明是抗微生物治疗的靶点。

DESCRIPTION (provided by applicant): RNA polymerase (RNAP) is the principal enzyme of gene expression and the target for genetic regulation. The long-term objective of this research is the understanding of the function of bacterial RNAP as a molecular machine at the atomic level of resolution. Specifically, the aims are (1) to build a model of the active center that would assign function to specific amino acid residues, nucleotides and metal ions in reactions of RNA synthesis and degradation and to interpret structurally the interrelationship between these reactions; (2) to characterize conformational transitions in the ternary transcribing complex that modulate catalytic function; (3) to understand transitions in the initial transcribing complex that take place during initial buildup of the nascent transcript, the release of the initiation factor sigma and promoter clearance; and (4) to explore the plasticity of RNAP molecule through generation of aptamers so that multiple conformations of RNAP could be captured for crystallographic studies. To these ends, a series of functionally defined complexes will be generated, in which RNAP will be (a) stalled at a particular stage of the transcription process; or (b) complexed with a defined nucleic acid scaffold, or (c) frozen in a complex with a bound aptamer. The complexes will be studied using chemical nucleic acid-protein crosslinks, genetically engineered mutations in RNAP, and discriminative biochemical assays. The results will be interpreted with the aid of molecular modeling. The understanding of the basic transcription mechanisms generated in this research is a prerequisite for molecular interpretation of regulatory phenomena and the development of interventions into gene expression. In addition, knowledge gained in these studies will be helpful for rational design and screening of new inhibitors of RNAP, which has been a proven target for anti-microbial therapy.