Diaz G A, Rong M, McAllister W T, Durbin R K
Morse Institute of Molecular Genetics, Department of Microbiology and Immunology, SUNY Health Science Center at Brooklyn 11203, USA.
Biochemistry. 1996 Aug 20;35(33):10837-43. doi: 10.1021/bi960488+.
We have developed a promoter competition assay to determine whether T7 RNA polymerase dissociates from its template during abortive cycling. We find that the stability of the initiation complex (IC) depends upon the conformation of the promoter, and that the degree to which the template is unwound contributes importantly to the stability of the IC. On linear DNA or a relaxed plasmid template, the stability of the IC is very low (t1/2 < 1 min). However, on a supercoiled template, the IC has a stability that is comparable to that of a paused elongation complex (t1/2 = 14 min). At a synthetic promoter that is single stranded in the initiation region (from -5 and downstream), the polymerase forms a highly stable complex (t1/2 > 30 min) even in the absence of RNA synthesis. These findings are important to our understanding of the transition from the IC to an EC.
我们开发了一种启动子竞争分析方法，以确定T7 RNA聚合酶在流产循环期间是否会从其模板上解离。我们发现起始复合物（IC）的稳定性取决于启动子的构象，并且模板解旋的程度对IC的稳定性有重要贡献。在线性DNA或松弛质粒模板上，IC的稳定性非常低（半衰期<1分钟）。然而，在超螺旋模板上，IC的稳定性与暂停延伸复合物相当（半衰期=14分钟）。在起始区域（从-5及下游）为单链的合成启动子处，即使在没有RNA合成的情况下，聚合酶也会形成高度稳定的复合物（半衰期>30分钟）。这些发现对于我们理解从IC到延伸复合物（EC）的转变非常重要。
