ACTIVE SITE & MECHANISM OF RNA POLYMERASE

这项研究的目的是提供关于 依赖DNA的RNA聚合酶的功能及其催化机制。至 为了获得这些信息，我们建议应用各种物理和 化学技术，包括吸收光谱、圆二色谱 (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的络合物、底物、底物类似物和特异性 抑制剂。我们打算研究的RNA聚合酶机制的几个方面 探讨了：(1)模板与RNA聚合酶的结合；(2)模板的性质和 酶中催化基团的定位；(3)引发和 伸长过程。1.模板绑定。这个世界的本质 DNA和蛋白质在形成过程中的构象变化 我们将探索封闭和开放的启动子位点。克隆限制 将使用含有启动子区域的片段。 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亚单位在DNA中的作用 将通过确定Sigma是否可能绑定到 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。酪氨酸残基的硝化将被 研究，特别是那些关于西格玛亚单位的研究。亲和力的场所 标记和亲光性标记将按链顺序确定 分离、裂解和多肽图谱。非共价光氧化 以及共价键合染料的研究。3.印心和 伸长率。NTPs和起始性二核苷酸与酶的结合 并对酶-DNA复合体进行了光谱研究。 含有特定启动子的片段将被转录成 明确定义的点，以检查启动之间的构象差异 和伸长复合体。

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.