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，也是基因调控的目标。这项研究的长期目标是在原子分辨率水平上理解细菌RNAP作为分子机器的功能。具体地说，其目的是(1)建立活性中心的模型，将功能分配给特定的氨基酸残基、核苷酸和金属离子在RNA合成和降解反应中，并从结构上解释这些反应之间的相互关系；(2)表征三元转录复合体中调节催化功能的构象转变；(3)了解初始转录复合体中发生的转变，这些转变发生在新生转录本的初始建立过程中，启动因子Sigma的释放和启动子的清除；以及(4)通过产生适配子来探索RNAP分子的可塑性，以便捕获多种构象的RNAP用于结晶学研究。为此，将产生一系列功能定义的复合体，其中RNAP将(A)在转录过程的特定阶段停滞；或(B)与定义的核酸支架络合，或(C)冻结在具有结合适体的复合体中。这些复合体将使用化学核酸-蛋白质交联物、RNAP基因工程突变和区别性生化分析进行研究。结果将借助分子模型进行解释。理解这项研究中产生的基本转录机制是对调控现象的分子解释和对基因表达干预的发展的先决条件。此外，在这些研究中获得的知识将有助于合理设计和筛选RNAP的新抑制剂，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.