Structure and Function of RNA Polymerases in E Coli

大肠杆菌 RNA 聚合酶的结构和功能

• 批准号: 7192412
• 负责人: ARKADY MUSTAEV
• 金额: $ 53.25万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7192412
• 关键词: Amino Acids; Binding; Biochemical; Biological Assay; Chemicals; Complex; Crystallization; DNA-Directed RNA Polymerase; Engineering; Enzyme Gene; Escherichia coli; Freezing; Gene Expression; Generations; Genetic; Genetic Transcription; Ions; Knowledge; Metals; Modeling; Molecular; Molecular Conformation; Molecular Machines; Mutation; Nucleic Acids; Nucleotides; Peptide Initiation Factors; RNA Polymerase Inhibitor; RNA chemical synthesis; Reaction; Recruitment Activity; Regulation; Research; Resolution; Screening procedure; Series; Sigma Factor; Staging; Structure; Transcript; Transcription Process; antimicrobial; aptamer; base; conformational conversion; design; molecular modeling; promoter; protein crosslink; scaffold; therapy development

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.

描述（由申请人提供）：RNA聚合酶（RNAP）是基因表达的主要酶，也是遗传调节的靶标。这项研究的长期目的是了解细菌RNAP在原子分辨率水平上作为分子机的功能。具体而言，目的是（1）建立一个活跃中心的模型，该模型将功能分配给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，并在结构上解释这些反应之间的相互关系； （2）表征在调节催化功能的三元转录复合物中的构象转变； （3）了解在最初堆积新生转录本期间发生的初始转录复合物中的过渡，请释放启动因子Sigma和启动子清除率； （4）通过生成适体探索RNAP分子的可塑性，以便可以捕获RNAP的多种构象进行晶体学研究。在这些目的上，将生成一系列功能定义的复合物，其中RNAP将（a）停滞在转录过程的特定阶段；或（b）与定义的核酸支架复合，或（c）在具有结合的适体的复合物中冷冻。将使用化学核酸 - 蛋白交联，RNAP中的基因工程突变和歧视性生化测定进行研究。结果将借助分子建模来解释。对本研究产生的基本转录机制的理解是对调节现象的分子解释的先决条件，并将干预措施发展为基因表达。此外，在这些研究中获得的知识将有助于理性的设计和筛选RNAP的新抑制剂，这已被证明是抗微生物疗法的靶标。