Landini P, Bown J A, Volkert M R, Busby S J
School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
J Biol Chem. 1998 May 22;273(21):13307-12. doi: 10.1074/jbc.273.21.13307.
The methylated form of the Ada protein (meAda) binds the ada and aidB promoters between 60 and 40 base pairs upstream from the transcription start and activates transcription of the Escherichia coli ada and aidB genes. This region is also a binding site for the alpha subunit of RNA polymerase and resembles the rrnB P1 UP element in A/T content and location relative to the core promoter. In this report, we show that deletion of the C-terminal domain of the alpha subunit severely decreases meAda-independent binding of RNA polymerase to ada and aidB, affecting transcription initiation at these promoters. We provide evidence that meAda activates transcription by direct interaction with the C-terminal domain of RNA polymerase sigma70 subunit (amino acids 574-613). Several negatively charged residues in the sigma70 C-terminal domain are important for transcription activation by meAda; in particular, a glutamic acid to valine substitution at position 575 has a dramatic effect on meAda-dependent transcription. Based on these observations, we propose that the role of the alpha subunit at ada and aidB is to allow initial binding of RNA polymerase to the promoters. However, transcription initiation is dependent on meAda-sigma70 interaction.
Ada蛋白的甲基化形式（meAda）在转录起始点上游60至40个碱基对之间结合ada和aidB启动子，并激活大肠杆菌ada和aidB基因的转录。该区域也是RNA聚合酶α亚基的结合位点，在A/T含量以及相对于核心启动子的位置上类似于rrnB P1上游元件。在本报告中，我们表明α亚基C末端结构域的缺失严重降低了RNA聚合酶与ada和aidB的非meAda依赖性结合，影响了这些启动子处的转录起始。我们提供证据表明，meAda通过与RNA聚合酶σ70亚基的C末端结构域（氨基酸574 - 613）直接相互作用来激活转录。σ70 C末端结构域中的几个带负电荷的残基对于meAda介导的转录激活很重要；特别是，第575位的谷氨酸被缬氨酸取代对依赖meAda的转录有显著影响。基于这些观察结果，我们提出α亚基在ada和aidB处的作用是允许RNA聚合酶最初结合到启动子上。然而，转录起始依赖于meAda - σ70相互作用。
