Monroe R S, Ostrowski J, Hryniewicz M M, Kredich N M
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
J Bacteriol. 1990 Dec;172(12):6919-29. doi: 10.1128/jb.172.12.6919-6929.1990.
The cysteine regulons of Salmonella typhimurium and Escherichia coli are positively regulated by CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, both of which act as inducers. Gel mobility shift assays and DNase I footprinting experiments showed that CysB protein binds to the S. typhimurium cysK promoter at two sites, one, designated CBS-K1, at positions -78 to -39 relative to the major transcription start site, and the other, designated CBS-K2, at positions -115 to -79. The S. typhimurium cysJIH promoter was found to contain a single binding site, designated CBS-JH, at positions -76 to -35. Acetyl-L-serine stimulated binding to CBS-K1 and CBS-J and inhibited binding to CBS-K2. In the absence of acetyl-L-serine, CysB protein bound to both CBS-K1 and CBS-K2 and gave a complex that migrated more slowly during gel electrophoresis than did that formed in the presence of acetyl-L-serine, in which case CysB protein bound only to CBS-K1. Complexes formed with DNA containing the two binding sites either at the middle or at one end of the fragment migrated differently, suggesting that DNA was bent in the slow complex formed in the absence of acetyl-L-serine and that DNA in the fast complex was less bent or not bent at all. An analysis of upstream deletions of the cysK promoter showed that only CBS-K1 is required for in vivo promoter activity. CBS-J is analogous in position to CBS-K1 and is probably also required for activity of the cysJIH promoter. CBS-K2 has no known function but may help sequester CysB protein at the cysK promoter.
鼠伤寒沙门氏菌和大肠杆菌的半胱氨酸调节子受CysB蛋白以及O-乙酰-L-丝氨酸或N-乙酰-L-丝氨酸的正向调节，二者均作为诱导剂。凝胶迁移率变动分析和DNase I足迹实验表明，CysB蛋白在两个位点与鼠伤寒沙门氏菌cysK启动子结合，一个位点命名为CBS-K1，位于相对于主要转录起始位点的-78至-39位，另一个位点命名为CBS-K2，位于-115至-79位。发现鼠伤寒沙门氏菌cysJIH启动子在-76至-35位含有一个单一结合位点，命名为CBS-JH。乙酰-L-丝氨酸刺激与CBS-K1和CBS-J的结合，并抑制与CBS-K2的结合。在没有乙酰-L-丝氨酸的情况下，CysB蛋白与CBS-K1和CBS-K2都结合，形成的复合物在凝胶电泳中迁移得比在有乙酰-L-丝氨酸存在时形成的复合物慢，在有乙酰-L-丝氨酸的情况下，CysB蛋白只与CBS-K1结合。与含有两个结合位点的DNA在片段中间或一端形成的复合物迁移方式不同，这表明在没有乙酰-L-丝氨酸时形成的慢迁移复合物中DNA发生了弯曲，而快迁移复合物中的DNA弯曲程度较小或根本没有弯曲。对cysK启动子上游缺失的分析表明，体内启动子活性只需要CBS-K1。CBS-J在位置上与CBS-K1类似，可能也是cysJIH启动子活性所必需的。CBS-K2没有已知功能，但可能有助于将CysB蛋白隔离在cysK启动子处。
