Structural Analysis of E.coli Transcription Complexes

大肠杆菌转录复合物的结构分析

• 批准号: 9020441
• 负责人: Michelle Hanna
• 金额: --
• 依托单位: University of California-Irvine
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-03-15 至 1991-07-01
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9020441&HistoricalAwards=false
• 关键词: Structural; Analysis; E.coli; Transcription; Complexes

The synthesis of RNA (transcription) is a process common to all organisms, and is catalyzed by the enzyme RNA polymerase. It is a central step in the flow of information from the DNA to cellular structure and function and is subject to considerable regulation. The transcription process can be divided into two types of activities. The first includes those steps which are common to all RNA polymerases: the synthesis of RNA by polymerization of nucleotides in the template - directed reaction. The second includes the regulatory processes which modulate transcription and differ in complexity and mechanism among organisms. The proposed experiments are designed to analyze these activities at the molecular level by employing photochemical crosslinking. The bacterium Escherichia coli will be used as a model for these studies. The in vitro transcription system employing purified E. coli RNA polymerase has been well characterized, and many transcriptional regulatory factors have been identified. By incorporating nucleotide analogs which are tagged with photoreactive crosslinking groups into the RNA during transcription, it is possible to probe the molecular environment of the RNA during its synthesis. RNA containing such analogs becomes covalently attached to adjacent molecules upon irradiation with ultraviolet light . RNA will be synthesized in vito which contains photocrosslinking groups at different positions within the RNA, and the contacts made between these groups and the RNA polymerase subunits and/or DNA template will be analyzed. The immediate goal of this work is to use photocrosslinking nucleotide analogs to identify the RNA polymerase domains involved in substrate and product binding, as well as the regions containing the catalytic activities for phosphodiester bond formation and RNA displacement from the DNA-RNA hybrid. These experiments should provide information about the fundamental mechanisms of RNA synthesis common to all RNA polymerases. The question conformational differences among transcription complexed from differentially regulated promoters (antiterminated, stringently controlled, etc.). The long range goal is to structurally characterize the E.coli transcription complex, understand the mechanism of RNA synthesis at the molecular level, and analyze the effects of regulatory factors such as NusA of ppGpp on RNA polymerase conformation and activity.

RNA的合成（转录）是所有生物共有的过程，并且由酶RNA聚合酶催化。 这是从DNA到细胞结构和功能的信息流的核心步骤，并且受到相当大的调节。 转录过程可以分为两种类型的活动。 第一个包括所有RNA聚合酶共有的步骤：通过模板定向反应中核苷酸聚合的聚合来合成RNA。 第二个包括调节转录的调节过程，并在生物体之间的复杂性和机制上有所不同。 提出的实验旨在通过使用光化学交联来分析分子水平的这些活性。 大肠杆菌将用作这些研究的模型。 采用纯化的大肠杆菌RNA聚合酶的体外转录系统已得到很好的特征，并且已经确定了许多转录调节因子。 通过在转录过程中掺入将光电反应交联基团标记到RNA中的核苷酸类似物，可以在其合成过程中探测RNA的分子环境。 用紫外线照射后，含有这种类似物的RNA共价连接到相邻分子上。 RNA将在Vito中合成，其中包含RNA内不同位置的光叠链链接组，并且将分析这些组之间的接触和RNA聚合酶亚基和/或DNA模板。 这项工作的直接目标是使用光链接核苷酸类似物来识别参与底物和产物结合的RNA聚合酶结构域，以及含有磷酸二酯键形成的催化活性的区域，以及来自DNA-RNA杂交的RNA位移。 这些实验应提供有关所有RNA聚合酶共有的RNA合成基本机制的信息。 转录之间的问题构象差异是从差异调节的启动子（抗授物，严格控制等）复合的。 远距离目标是在结构上表征大肠杆菌转录复合物，了解分子水平上RNA合成的机理，并分析调节因子（例如PPGPP NUSA）对RNA聚合酶构象和活性的影响。