ACTIVE SITE & MECHANISM OF RNA POLYMERASE

这项研究的目的是提供新的信息， DNA依赖的RNA聚合酶的功能和催化机制。 到 为了获得这些信息，我们建议采用各种物理和 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 改性 这些方法将应用于RNA聚合酶本身， 其与DNA、底物、底物类似物和特异性 抑制剂的 我们打算对RNA聚合酶的作用机制作一综述。 探讨了模板与RNA聚合酶的结合情况， 催化基团在酶中的位置;（3）起始和 伸长过程 1. 模板的绑定。 的性质 DNA和蛋白质的构象变化与形成 将探索封闭和开放启动子位点。 克隆限制 将使用含启动子区域的片段。 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是否可能与 单链DNA 2. 催化基团的性质和位置。 化学改性研究的目的是确定哪些群体 参与催化。 酪氨酸残基的硝化将是 研究，特别是那些对西格玛亚基。 亲和位点 标记和光亲和标记将在序列中通过链确定 分离切割和肽图谱。 非共价光氧化 和共价连接的染料。 3. 起始和 伸长率 NTPs和起始二核苷酸与酶的结合 和酶-DNA复合物将通过光谱方法进行研究。 含有特异性启动子的片段将被转录成 定义明确的点，以检查起始之间的构象差异 和伸长复合物。

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.