Tapias Angels, Fernández Silvia, Alonso Juan C, Barbé Jordi
Departamento de Genética y Microbiología, Universitat Autónoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.
Nucleic Acids Res. 2002 Apr 1;30(7):1539-46. doi: 10.1093/nar/30.7.1539.
Transcription of the Rhodobacter sphaeroides recA promoter (P(recA)) is induced upon DNA damage in a lexA-dependent manner. In vivo experiments demonstrate that LexA protein represses and might also activate transcription of P(recA). Purified R.sphaeroides LexA protein specifically binds the SOS boxes located within the P(recA) region. In vitro transcription analysis, using Escherichia coli RNA polymerase (RNAP), indicated that the presence of LexA may stimulate and repress transcription of P(recA). EMSA and DNase I footprinting experiments show that LexA and RNAP can bind simultaneously to P(recA). At low LexA concentrations it enhances RNAP binding to P(recA), stimulates open complex formation and strand separation beyond the transcription start site. At high LexA concentrations, however, RNAP-promoted strand separation is not observed beyond the +5 region. LexA might repress transcription by interfering with the clearance process instead of blocking the access of RNAP to the promoter region. Based on these findings we propose that the R.sphaeroides LexA protein performs fine tuning of the SOS response, which might provide a physiological advantage by enhancing transcription of SOS genes and delaying full activation of the response.
球形红杆菌recA启动子(P(recA))的转录在DNA损伤时以LexA依赖的方式被诱导。体内实验表明LexA蛋白抑制并可能还激活P(recA)的转录。纯化的球形红杆菌LexA蛋白特异性结合位于P(recA)区域内的SOS框。使用大肠杆菌RNA聚合酶(RNAP)进行的体外转录分析表明,LexA的存在可能刺激和抑制P(recA)的转录。电泳迁移率变动分析(EMSA)和DNase I足迹实验表明LexA和RNAP可以同时结合到P(recA)。在低LexA浓度下,它增强RNAP与P(recA)的结合,刺激开放复合物形成以及转录起始位点以外的链分离。然而,在高LexA浓度下,在+5区域以外未观察到RNAP促进的链分离。LexA可能通过干扰清除过程而不是阻止RNAP进入启动子区域来抑制转录。基于这些发现,我们提出球形红杆菌LexA蛋白对SOS反应进行微调,这可能通过增强SOS基因的转录和延迟反应的完全激活而提供生理优势。