McClure W R, Cech C L, Johnston D E
J Biol Chem. 1978 Dec 25;253(24):8941-8.
A new assay yielding mechanistic information on the initiation reaction of Escherichia coli RNA polymerase has been developed. It was found to be useful in characterizing the promoters of bacteriophage DNA templates. The binding of the first two triphosphates in an RNA sequence was determined to be equilibrium ordered with ATP binding first followed by UTP on the lambda promoters PL. and PR. The products resulting from phosphodiester bond formation, pppApU and PPi, dissociated rapidly in the absence of the other triphosphates required for RNA synthesis. The resulting steady state conversion of ATP and UTP into pppApU was the basis for the new assay. The rate-limiting step in the initiation reaction was not precisely determined, but it was argued not to be entirely the release of product. The Zn2+ chelator, 1,10-phenanthroline, was partially characterized and found to be an uncompetitive inhibitor of ATP in the reaction (Ki = 100 micrometer). The unique advantage of this steady state assay is that several steps in the RNA initiation process are amplified kinetically and thus can be examined separately with techniques applicable to any other two-substrate, two-product enzyme reaction.
一种能提供有关大肠杆菌RNA聚合酶起始反应机制信息的新检测方法已被开发出来。发现它可用于表征噬菌体DNA模板的启动子。在λ噬菌体启动子PL和PR上，RNA序列中前两个三磷酸的结合被确定为平衡有序，首先是ATP结合，随后是UTP结合。磷酸二酯键形成产生的产物pppApU和PPi，在缺乏RNA合成所需的其他三磷酸时会迅速解离。ATP和UTP转化为pppApU的稳态转化是新检测方法的基础。起始反应中的限速步骤没有精确确定，但有人认为不完全是产物的释放。锌离子螯合剂1,10-菲咯啉已得到部分表征，发现它是该反应中ATP的非竞争性抑制剂（Ki = 100微摩尔）。这种稳态检测方法的独特优势在于，RNA起始过程中的几个步骤在动力学上被放大，因此可以用适用于任何其他双底物、双产物酶反应的技术分别进行研究。
