Rao L, Karls R K, Betley M J
Department of Bacteriology, University of Wisconsin-Madison 53706, USA.
J Bacteriol. 1995 May;177(10):2609-14. doi: 10.1128/jb.177.10.2609-2614.1995.
The RNA polymerase (RNAP) holoenzyme of Staphylococcus aureus was purified by DNA affinity, gel filtration, and ion-exchange chromatography. This RNAP contained four major subunits with apparent molecular masses of 165, 130, 60, and 47 kDa. All four subunits of the RNAP were serologically related to the subunits of Escherichia coli E sigma 70 holoenzyme by Western immunoblot analysis. The 60-kDa subunit was subsequently isolated and found to react with a monoclonal antibody specific to the E. coli sigma 70 subunit. This sigma 70-related protein allowed E. coli core RNAP promoter-specific initiation and increased transcription by S. aureus RNAP that is unsaturated with sigma. We therefore suggest that this 60-kDa protein is a sigma factor. Purified S. aureus RNAP transcribed from the promoters of several important S. aureus virulence genes (sea, sec, hla, and agr P2) in vitro. The in vitro transcription start sites of the sea, sec, and agr P2 promoters, mapped by primer extension, were similar to those identified in vivo. The putative promoter hexamers of these three genes showed strong sequence similarity to the E. coli sigma 70 consensus promoter, and transcription by E sigma 70 from some of these promoters has been observed. Conversely, S. aureus RNAP does not transcribe from all E. coli sigma 70-dependent promoters. Taken together, our results indicate that the promoter sequences recognized by purified S. aureus RNAP are similar but not identical to those recognized by E. coli E sigma 70.
通过DNA亲和层析、凝胶过滤和离子交换层析法纯化了金黄色葡萄球菌的RNA聚合酶（RNAP）全酶。这种RNAP包含四个主要亚基，其表观分子量分别为165、130、60和47 kDa。通过Western免疫印迹分析，RNAP的所有四个亚基在血清学上都与大肠杆菌E σ70全酶的亚基相关。随后分离出60 kDa的亚基，发现它能与一种针对大肠杆菌σ70亚基的单克隆抗体发生反应。这种与σ70相关的蛋白质使得大肠杆菌核心RNAP能够进行启动子特异性起始，并增加了未被σ饱和的金黄色葡萄球菌RNAP的转录。因此，我们认为这种60 kDa的蛋白质是一种σ因子。纯化的金黄色葡萄球菌RNAP在体外从几个重要的金黄色葡萄球菌毒力基因（sea、sec、hla和agr P2）的启动子进行转录。通过引物延伸法确定的sea、sec和agr P2启动子的体外转录起始位点与体内鉴定的位点相似。这三个基因的推定启动子六聚体与大肠杆菌σ70共有启动子表现出很强的序列相似性，并且已经观察到大肠杆菌E σ70从其中一些启动子进行转录。相反，金黄色葡萄球菌RNAP并非从所有大肠杆菌σ70依赖的启动子进行转录。综上所述，我们的结果表明，纯化的金黄色葡萄球菌RNAP识别的启动子序列与大肠杆菌E σ70识别的启动子序列相似但并不相同。
