Sterba Kristen M, Mackintosh Samuel G, Blevins Jon S, Hurlburt Barry K, Smeltzer Mark S
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
J Bacteriol. 2003 Aug;185(15):4410-7. doi: 10.1128/JB.185.15.4410-4417.2003.
The staphylococcal accessory regulator locus (sarA) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and cis elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene cna. The results suggest the existence of multiple high-affinity binding sites within the cna promoter region. Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including cna, fnbA, and sspA. The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the Staphylococcus aureus genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. By using the same criteria, consensus binding sites were also identified upstream of the S. aureus genes spa, fnbA, sspA, agr, hla, and cna. With the exception of cna, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene.
葡萄球菌辅助调节基因座（sarA）编码一种DNA结合蛋白（SarA），该蛋白可调节100多个基因的表达。目前尚不清楚这是否通过SarA与其靶基因相关的顺式元件之间的直接相互作用发生，部分原因是尚未确定SarA结合位点的明确特征。在这项研究中，采用电泳迁移率变动分析（EMSA）来鉴定SarA调节基因cna上游的SarA结合位点。结果表明在cna启动子区域存在多个高亲和力结合位点。我们还使用SELEX（指数富集配体系统进化）程序和纯化的重组SarA，从随机的DNA片段库中筛选出含有高亲和力SarA结合位点的DNA靶标。随后对这些片段进行克隆和测序。还通过EMSA对随机选择的克隆进行了检测。这些DNA片段与SarA的结合亲和力与公认的SarA调节基因（包括cna、fnbA和sspA）相当。SarA选择的DNA富含AT，这与金黄色葡萄球菌基因组的核苷酸组成一致。对所选DNA的比对揭示了一个7碱基的共有序列（ATTTTAT），在56个测序克隆中的46个中，该序列的错配不超过一个。按照相同标准，在金黄色葡萄球菌基因spa、fnbA、sspA、agr、hla和cna的上游也鉴定出了共有结合位点。除了之前未检测过结合位点的cna外，这个7碱基基序位于之前与每个基因相关的假定SarA结合位点内。
