大肠杆菌RNA聚合酶的两个α亚基呈不对称排列，并与DNA上游元件的不同半区接触。
The two alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element.
作者信息
Murakami K, Kimura M, Owens J T, Meares C F, Ishihama A
机构信息
Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan.
出版信息
Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1709-14. doi: 10.1073/pnas.94.5.1709.
Abstract
RNA polymerase core enzyme of Escherichia coli is composed of two alpha subunits and one each of the beta and beta' subunits. The C-terminal domain of the RNA polymerase alpha subunit plays a key role in molecular communications with class I transcription factors and upstream (UP) elements of promoter DNA, using the same protein surface. To identify possible differences in the functional roles of the two alpha subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct alpha subunit derivatives in a defined orientation ("oriented alpha-heterodimer"). The binding sites of two alpha C-terminal domains on the UP element DNA were determined by hydroxyl radical-based DNA cleavage mediated by (p-bromoacetamidobenzyl)-EDTA x Fe, which was bound at Cys-269 on the UP recognition surface of one or both alpha subunits. The results clearly indicated that the two alpha subunits bind in tandem to two helix turns of the rrnBP1 UP element, and that the beta'-associated alpha subunit is bound to the promoter-distal region.
摘要
大肠杆菌的RNA聚合酶核心酶由两个α亚基以及各一个β和β'亚基组成。RNA聚合酶α亚基的C末端结构域在与I类转录因子和启动子DNA的上游（UP）元件进行分子通讯时发挥关键作用，且使用相同的蛋白质表面。为了确定两个α亚基在功能作用上可能存在的差异，我们开发了一种重组方法，用于构建在特定方向上含有两种不同α亚基衍生物的杂交RNA聚合酶（“定向α-异二聚体”）。通过由（对溴乙酰氨基苄基）-EDTA·Fe介导的基于羟基自由基的DNA切割，确定了两个α C末端结构域在UP元件DNA上的结合位点，该物质结合在一个或两个α亚基的UP识别表面的半胱氨酸-269位点上。结果清楚地表明，两个α亚基串联结合到rrnBP1 UP元件的两个螺旋圈上，并且与β'相关的α亚基结合到启动子远端区域。
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