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，也是基因调控的靶标。这项研究的长期目标是了解细菌RNAP作为分子机器在原子水平上的功能。具体而言，目标是：(1)建立一个活性中心模型，该模型将在RNA合成和降解反应中为特定的氨基酸残基、核苷酸和金属离子分配功能，并从结构上解释这些反应之间的相互关系；(2)表征三元转录复合物中调节催化功能的构象转变；(3)了解在新生转录物的初始积累、起始因子sigma的释放和启动子清除过程中发生的初始转录复合物的转变；(4)通过生成适配体来探索RNAP分子的可塑性，从而捕获RNAP的多种构象进行晶体学研究。为此，将产生一系列功能定义的复合物，其中RNAP将(a)在转录过程的特定阶段停滞；或(b)与确定的核酸支架络合，或(c)与结合的适体在络合物中冷冻。这些复合物将通过化学核酸-蛋白交联、RNAP基因工程突变和鉴别生化分析进行研究。这些结果将借助分子模型加以解释。本研究中产生的基本转录机制的理解是分子解释调控现象和发展干预基因表达的先决条件。此外，这些研究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.