Chien Y, Cheung A L
Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, New York 10021, USA.
J Biol Chem. 1998 Jan 30;273(5):2645-52. doi: 10.1074/jbc.273.5.2645.
The expression of many virulence determinants in Staphylococcus aureus is controlled by regulatory loci such as agr and sar. We have previously shown that the SarA protein is required for optimal transcription of RNAII and RNAIII in the agr locus. To define the specific molecular interaction, we overexpressed SarA as a glutathione S-transferase (GST) fusion protein by cloning the 372-base pair (bp) sarA gene into the vector. The purified GST-SarA as well as cleaved SarA were able to bind specifically to the P2, P3, and the combined P2-P3 promoter fragments of agr in gel shift assays. Using monoclonal antibodies to SarA, we found that SarA is a part of the retarded protein-DNA complex as evidenced by the formation of a supershifted band. The SarA binding site on the agr promoter, mapped by DNase I footprinting assay, covered a 29-bp region between the P2 and P3 promoters devoid of any direct repeats. A synthetic 45-bp fragment encompassing the 29-bp sequence also bound the SarA protein in band shift assays. Serial in-frame deletion analysis of sarA revealed that, with the exception of 15 residues in the N terminus, almost all of SarA (residues 16-124) is essential for agr binding activity. Northern analysis confirmed that only the sar mutant clone containing a truncated sarA gene with a 15-residue deletion in the N terminus (SarA16-124) could activate agr transcription to a level approaching that of the full-length counterpart (SarA1-124). Taken together, these data indicated that SarA is a DNA-binding protein with binding specificity to the P2 and P3 interpromoter region of agr, thereby activating RNAII and RNAIII transcription.
金黄色葡萄球菌中许多毒力决定因素的表达受诸如agr和sar等调控位点的控制。我们先前已表明，SarA蛋白是agr位点中RNAII和RNAIII最佳转录所必需的。为了确定具体的分子相互作用，我们通过将372个碱基对（bp）的sarA基因克隆到载体中，以谷胱甘肽S-转移酶（GST）融合蛋白的形式过表达SarA。在凝胶迁移试验中，纯化的GST-SarA以及切割后的SarA能够特异性结合agr的P2、P3以及组合的P2-P3启动子片段。使用针对SarA的单克隆抗体，我们发现SarA是延迟的蛋白质-DNA复合物的一部分，超迁移带的形成证明了这一点。通过DNase I足迹试验绘制的agr启动子上的SarA结合位点，覆盖了P2和P3启动子之间一个没有任何直接重复序列的29 bp区域。在带迁移试验中，一个包含29 bp序列的合成45 bp片段也能结合SarA蛋白。sarA的连续读框内缺失分析表明，除了N端的15个残基外，几乎所有的SarA（第16 - 124位残基）对于agr结合活性都是必需的。Northern分析证实，只有包含在N端有15个残基缺失的截短sarA基因的sar突变克隆（SarA16 - 124）能够将agr转录激活到接近全长对应物（SarA1 - 124）的水平。综上所述，这些数据表明SarA是一种对agr的P2和P3启动子间区域具有结合特异性的DNA结合蛋白，从而激活RNAII和RNAIII转录。
