ACTIVE SITE & MECHANISM OF RNA POLYMERASE

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• 批准号: 3271844
• 负责人: ROBERT W WOODY
• 金额: $ 13.92万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-06-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271844
• 关键词: DNA; DNA directed RNA polymerase; Escherichia coli; affinity labeling; bacterial proteins; bacterial virus; chemical group; circular dichroism; conformation; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate complex; fluorescence spectrometry; fluorescent dye /probe; genetic promoter element; genetic transcription; microorganism genetics; molecular cloning; molecular rearrangement; nitrification; nucleic acid structure; photochemistry; protein biosynthesis; radiotracer; spectrometry; stop flow technique; tyrosine; virus DNA

The objective of this research is to provide new information about the function and catalytic mechanism of DNA-dependent RNA polymerase. To obtain such information, we propose to apply a variety of physical and chemical techniques, including absorption spectroscopy, circular dichroism (CD), fluorescence, stopped-flow kinetics, chemical and photochemical modification. These methods will be applied to RNA polymerase itself and to its complexes with DNA, substrates, substrate analogs and specific inhibitors. The aspects of RNA polymerase mechanism which we intend to explore are: (1) Binding of template to RNA polymerase; (2) The nature and location of catalytic groups in the enzyme; (3) The initiation and elongation process. 1. Binding of template. The nature of the conformational changes in the DNA and protein associated with formation of both closed and open promoter sites will be explored. Cloned restriction fragments of promoter-containing regions will be used. Fluorescence-labeled RNA polymerase will be used in stopped-flow kinetics studies of DNA binding to polymerase. The role of the Sigma subunit in DNA binding will be tested by determining whether Sigma might bind to single-stranded DNA. 2. Nature and location of catalytic groups. Chemical modification studies will be aimed at determining what groups participate in the catalysis. Nitration of tyrosine residues will be studied, especially those on the Sigma subunit. The site of affinity labels and photoaffinity labels will be determined in the sequence by chain separation cleavage and peptide mapping. Photooxidation with noncovalently and covalently attached dyes will be studied. 3. Initiation and elongation. The binding of NTPs and initiating dinucleotides to the enzyme and to enzyme-DNA complexes will be studied by spectroscopic methods. Specific promoter-containing fragments will be transcribed to a well-defined point to examine conformational differences between initiation and elongation complexes.

这项研究的目的是提供有关 DNA依赖性RNA聚合酶的功能和催化机制。 到 获得此类信息，我们建议应用各种物理和 化学技术，包括吸收光谱、圆二色性 (CD)、荧光、停流动力学、化学和光化学 修改。 这些方法将应用于RNA聚合酶本身和 其与 DNA、底物、底物类似物和特定物质的复合物 抑制剂。 我们打算研究的RNA聚合酶机制的各个方面 探索的是：（1）模板与RNA聚合酶的结合； (2) 性质及 酶中催化基团的位置； (3)发起和 伸长过程。 1、模板的装订。 的性质 与形成相关的DNA和蛋白质的构象变化 将探索封闭和开放的启动子站点。 克隆限制 将使用含有启动子的区域的片段。 荧光标记的 RNA 聚合酶将用于停流动力学 DNA 与聚合酶结合的研究。 Sigma 亚基在 DNA 中的作用 将通过确定 Sigma 是否可以结合来测试结合 单链DNA。 2.催化基团的性质和位置。 化学修饰研究的目的是确定哪些基团 参与催化。 酪氨酸残基将被硝化 研究，特别是那些关于西格玛亚基的研究。 亲和位点 标签和光亲和标签将按链顺序确定 分离裂解和肽图谱。 非共价光氧化 并将研究共价连接的染料。 3. 发起和 伸长。 NTP 和起始二核苷酸与酶的结合 将通过光谱方法研究酶-DNA 复合物。 含有特定启动子的片段将被转录成 明确的点来检查起始之间的构象差异 和伸长复合物。