THE CYSP PROMOTER OF SALMONELLA-TYPHIMURIUM - CHARACTERIZATION OF 2 BINDING-SITES FOR CYSB PROTEIN, STUDIES OF INVIVO TRANSCRIPTION INITIATION, AND DEMONSTRATION OF THE ANTI-INDUCER EFFECTS OF THIOSULFATE
THE CYSP PROMOTER OF SALMONELLA-TYPHIMURIUM - CHARACTERIZATION OF 2 BINDING-SITES FOR CYSB PROTEIN, STUDIES OF INVIVO TRANSCRIPTION INITIATION, AND DEMONSTRATION OF THE ANTI-INDUCER EFFECTS OF THIOSULFATE
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DOI：
10.1128/jb.173.18.5876-5886.1991
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发表时间：
1991-09-01
影响因子：
3.2
通讯作者：
KREDICH, NM
中科院分区：
文献类型：
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作者：
HRYNIEWICZ, MM;KREDICH, NM
大肠杆菌和鼠伤寒沙门氏菌的Cysptwa操纵子编码硫酸盐和硫代硫酸盐的周质传输系统的成分，并作为半胱氨酸调节剂的一部分进行调节。 从CYSP启动子发起的体外转录起始则表明需要CysB蛋白和O-乙酰L-丝氨酸或N-乙酰L-丝氨酸，它们是诱导剂，并被抗诱导剂硫化物抑制。 发现硫代硫酸盐比作为抗诱导剂的硫化物更有效。 DNase I保护实验显示在存在N-乙酰L-丝氨酸的情况下，CYSB蛋白的两个离散结合位点。 CBS -P1位于-85和-41的位置之间，相对于主要的转录起始位点，CBS -P2位于-19和+25位置之间。 没有N-乙酰L-丝氨酸，CYSB蛋白保护位置-63和-11之间的区域，该区域被指定为CBS-P3。 在凝胶迁移率转移测定中，O-乙酰L-丝氨酸增加了CYSB蛋白-Cysp启动子复合物的迁移率。 N-乙酰基-L-丝氨酸在凝胶移位实验中无效，大概是因为其阴离子电荷导致其在电泳过程中从复合物中迅速去除。 相对于结合位点不同的DNA片段的比较表明，与Cysb蛋白的复合物包含在CBS-P1和CBS-P2之间弯曲的DNA，并且O-乙酰基-L-丝氨酸可降低DNA弯曲。 与单独包含CBS-P2，单独CBS-P1或整个CYSP启动子区域的片段结合研究，提出了一个模型，其中在没有O-乙酰基-L丝氨酸的情况下观察到的弯曲DNA的复合物包含单个Cysb蛋白分子绑定到CBS-P3。 在相对较低的CYSB蛋白浓度下，O-乙酰基-L-丝氨酸会导致单个CYSB蛋白分子与CBS-P1紧密结合，而不是与CBS-P3结合，从而降低DNA弯曲并增加复杂的电泳迁移率。 在较高的CYSB蛋白浓度下，O-乙酰基-L-丝氨酸会导致第二个分子在CBS-P2处结合，从而使迁移较慢。
The cysPTWA operons of Escherichia coli and Salmonella typhimurium encode components of periplasmic transport systems for sulfate and thiosulfate and are regulated as part of the cysteine regulons. In vitro transcription initiation from the cysP promoter was shown to require both CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, which act as inducers, and was inhibited by the anti-inducer sulfide. Thiosulfate was found to be even more potent than sulfide as an anti-inducer. DNase I protection experiments showed two discrete binding sites for CysB protein in the presence of N-acetyl-L-serine. CBS-P1 is located between positions -85 and -41 relative to the major transcription start site, and CBS-P2 is located between positions -19 and +25. Without N-acetyl-L-serine, the CysB protein protected the region between positions -63 and -11, which was designated CBS-P3. In gel mobility shift assays, the mobility of CysB protein-cysP promoter complexes was increased by O-acetyl-L-serine. N-Acetyl-L-serine had no effect in gel shift experiments, presumably because its anionic charge results in its rapid removal from the complex during electrophoresis. Comparison of DNA fragments differing with respect to binding site position indicated that complexes with CysB protein contain DNA that is bent somewhere between CBS-P1 and CBS-P2 and that O-acetyl-L-serine decreases DNA bending. Binding studies with fragments containing either CBS-P2 alone, CBS-P1 alone, or the entire cysP promoter region suggest a model in which the complex of bent DNA observed in the absence of O-acetyl-L-serine contains a single CysB protein molecule bound to CBS-P3. At relatively low CysB protein concentrations, O-acetyl-L-serine would cause a single CysB protein molecule to bind tightly to CBS-P1, rather than to CBS-P3, thereby decreasing DNA bending and increasing complex electrophoretic mobility. At higher CysB protein concentrations, O-acetyl-L-serine would cause a second molecule to bind at CBS-P2, giving a more slowly migrating complex.