DataSheet1_Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein.PDF

Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158.

RNA聚合酶识别启动子是基因表达调控的关键环节。细菌RNA聚合酶核心酶由五个亚基组成，可与一组σ因子（sigma factor）中的一员瞬时结合，进而形成RNA聚合酶全酶。σ因子可赋予RNA聚合酶启动子识别特异性。在革兰氏阳性致病菌肺炎链球菌（Streptococcus pneumoniae）中，大多数启动子大概率由SigA识别——SigA是一种研究尚不充分的持家σ因子（housekeeping sigma factor）。本研究通过序列保守性分析，发现SigA与大肠杆菌（Escherichia coli）和枯草芽孢杆菌（Bacillus subtilis）的同源蛋白具有相似的蛋白质结构：其N端100个氨基酸残基保守性较低，而蛋白其余区域（结构域2、3、4）保守性良好。此外，我们纯化了肺炎链球菌R6菌株编码的天然（无标签）SigA蛋白，并重构了由大肠杆菌核心酶与σ因子SigA组成的RNA聚合酶全酶（RNAP-SigA）。通过体外转录实验，我们发现RNAP-SigA不仅能够识别肺炎链球菌染色体上的特异性启动子，还能识别无乳链球菌（S. agalactiae）的广谱耐药质粒pMV158上的启动子。具体而言，SigA可引导RNA聚合酶转录质粒pMV158中参与复制与结合转移的基因。本研究结果表明，SigA在启动子识别方面具有多功能性，且其对质粒pMV158的广谱宿主适应性具有重要贡献。