Colland F, Orsini G, Brody E N, Buc H, Kolb A
Unité de Physicochimie des Macromolécules Biologiques, URA 1149 du CNRS, Institut Pasteur, Paris, France.
Mol Microbiol. 1998 Feb;27(4):819-29. doi: 10.1046/j.1365-2958.1998.00729.x.
The AsiA protein, encoded by bacteriophage T4, inhibits Esigma70-dependent transcription at bacterial and early-phage promoters. We demonstrate that the inhibitory action of AsiA involves interference with the recognition of the -35 consensus promoter sequence by host RNA polymerase. In vitro experiments were performed with a C-terminally labelled sigma factor that is competent for functional holoenzyme reconstitution. By protease and hydroxyl radical protein footprinting, we show that AsiA binds region 4.2 of sigma70, which recognizes the -35 sequence. Direct interference with the recognition of the promoter at this locus is supported by two parallel experiments. The stationary-phase sigma factor containing holoenzyme, which can initiate transcription at promoters devoid of a -35 region, is insensitive to AsiA inhibition. The recognition of a galP1 promoter by Esigma70 is not affected by the presence of AsiA. Therefore, we conclude that AsiA inhibits transcription from Escherichia coli and T4 early promoters by counteracting the recognition of region 4.2 of sigma70 with the -35 hexamer.
由噬菌体T4编码的AsiA蛋白可抑制细菌和早期噬菌体启动子处依赖于σ70的转录。我们证明，AsiA的抑制作用涉及干扰宿主RNA聚合酶对-35共有启动子序列的识别。使用能够进行功能性全酶重构的C末端标记的σ因子进行了体外实验。通过蛋白酶和羟基自由基蛋白足迹法，我们表明AsiA结合σ70的4.2区域，该区域识别-35序列。两个平行实验支持在该位点对启动子识别的直接干扰。含有全酶的稳定期σ因子可在没有-35区域的启动子处启动转录，对AsiA抑制不敏感。σ70对galP1启动子的识别不受AsiA存在的影响。因此，我们得出结论，AsiA通过抵消σ70的4.2区域与-35六聚体的识别来抑制大肠杆菌和T4早期启动子的转录。
