Luse D S, Kochel T, Kuempel E D, Coppola J A, Cai H
J Biol Chem. 1987 Jan 5;262(1):289-97.
We have prepared RNA polymerase II preinitiation complexes by incubating templates containing the adenovirus 2 major late promoter with HeLa cell nuclear extracts in the absence of nucleoside triphosphates. These preinitiation complexes are partially purified by gel filtration and are then provided with the appropriate substrates to allow either one or two phosphodiester bonds to be formed. When substrates that allow only one bond to form are used, no stable ternary complex is obtained and no RNA is made that can be incorporated into longer RNA chains. A stable complex is obtained, however, if the RNA polymerase can make two bonds. The production of a stable ternary complex requires ATP or dATP and is inhibited by alpha-amanitin. In the course of exploring the energy requirement for initiation we found that dATP may be incorporated, in the absence of ATP, as the initial base of the RNA. However, deoxyribonucleotides are not appreciably incorporated into the body of the transcript after the first two bases have been added to the growing chain.
我们通过在无核苷三磷酸的情况下，将含有腺病毒2主要晚期启动子的模板与HeLa细胞核提取物一起孵育，制备了RNA聚合酶II预起始复合物。这些预起始复合物通过凝胶过滤进行部分纯化，然后提供适当的底物以允许形成一个或两个磷酸二酯键。当使用仅允许形成一个键的底物时，不会获得稳定的三元复合物，也不会产生可掺入更长RNA链中的RNA。然而，如果RNA聚合酶可以形成两个键，则会获得稳定的复合物。稳定三元复合物的产生需要ATP或dATP，并受到α-鹅膏蕈碱的抑制。在探索起始的能量需求过程中，我们发现，在没有ATP的情况下，dATP可能作为RNA的初始碱基被掺入。然而，在前两个碱基添加到生长链后，脱氧核糖核苷酸不会明显掺入转录本主体中。
