Nomura T, Fujita N, Ishihama A
Department of Molecular Genetics, National Institute of Genetics, and School of Life Science, Graduate University for Advances Studies, Mishima, Shizuoka 411-8540, Japan.
Biochemistry. 1999 Jan 26;38(4):1346-55. doi: 10.1021/bi982381n.
The RNA polymerase core enzyme of Escherichia coli is composed of 2alpha, 1beta, and 1beta' subunits. Previously we mapped the alpha-alpha, alpha-beta, and alpha-beta' contact sites on the alpha subunit. Here we analyzed the alpha subunit contact sites on the beta subunit by using various experimental approaches: (i) comparison of the proteolytic cleavage map between the unassembled free beta subunit and the alpha2 beta complex; (ii) analysis of the binary complex formation between His6-tagged intact alpha subunit and various truncated beta fragments; and (iii) analysis of the complex formation between the alpha subunit and various His6-tagged beta fragments. The results altogether indicate that two regions of the beta subunit are involved in the full activity of alpha binding, that is, the primary contact site between residues 737 and 904 and the secondary region with assembly control activity downstream from residue 1138. All of the alpha subunit-beta fragment binary complexes identified in this study were found to bind beta' subunit and form pseudo-core complexes, indicating that the regions of beta involved in alpha subunit contact also participate in interaction with the beta' subunit.
大肠杆菌的RNA聚合酶核心酶由2个α亚基、1个β亚基和1个β'亚基组成。此前我们已在α亚基上绘制了α-α、α-β和α-β'的接触位点。在此，我们通过多种实验方法分析了β亚基上的α亚基接触位点：（i）比较未组装的游离β亚基与α2β复合物之间的蛋白水解图谱；（ii）分析His6标记的完整α亚基与各种截短的β片段之间的二元复合物形成；以及（iii）分析α亚基与各种His6标记的β片段之间的复合物形成。结果共同表明，β亚基的两个区域参与了α结合的全部活性，即737至904位残基之间的主要接触位点以及1138位残基下游具有组装控制活性的次要区域。本研究中鉴定的所有α亚基-β片段二元复合物均被发现可结合β'亚基并形成假核心复合物，这表明β中参与α亚基接触的区域也参与了与β'亚基的相互作用。
