Colyer T E, Kredich N M
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Mol Microbiol. 1996 Jul;21(2):247-56. doi: 10.1046/j.1365-2958.1996.6301347.x.
Expression of the cysteine regulon in Salmonella typhimurium and Escherichia coli is controlled by the LysR-type transcriptional activator CysB and by the inducer N-acetyl-L-serine. Sulphide and thiosulphate are anti-inducers. Two highly purified constitutive CysB proteins, CysB(T149M) and CysB(T149P), were found to bind to the cysJIH, cysK and cysP promoters, to activate transcription from the cysJIH and cysK promoters in the absence of N-acetyl-L-serine, and to be insensitive to the effects of anti-inducers. At 10 mM MgCl2, the in vitro transcription activity of CysB(T149M) was maximal without N-acetyl-L-serine, but that of CysB(T149P) was increased by inducer. At 2 mM MgCl2, both proteins were fully active without inducer. A third mutant protein, CysB(W166R), was totally inactive at 10 mM MgCl2, but gave constitutive expression of the cysK and cysJIH promoters at 2 mM MgCl2. Surprisingly, wild-type CysB was also constitutive for the cysK promoter at 2 mM mgCl2 but not at 10 mM MgCl2; it required inducer for cysJIH promoter activation at both concentrations. Mutagenic studies indicated that this difference between promoters is due to the distance between activation site half-sites, which are separated by 1 bp in the cysJIH promoter and by 2 bp in the cysK promoter. We speculate that inducer acts to decrease the distance between the binding domains of two CysB subunits that interact with an activation site. In vitro activities of wild-type and mutant CysB proteins correlated much better with in vivo behaviour at 2 mM than at 10 mM MgCl2, suggesting that the former is the more physiological concentration.
鼠伤寒沙门氏菌和大肠杆菌中半胱氨酸调节子的表达受LysR型转录激活因子CysB和诱导剂N-乙酰-L-丝氨酸的控制。硫化物和硫代硫酸盐是抗诱导剂。发现两种高度纯化的组成型CysB蛋白，即CysB(T149M)和CysB(T149P)，能与cysJIH、cysK和cysP启动子结合，在没有N-乙酰-L-丝氨酸的情况下激活cysJIH和cysK启动子上的转录，并且对抗诱导剂的作用不敏感。在10 mM MgCl2条件下，CysB(T149M)的体外转录活性在没有N-乙酰-L-丝氨酸时最大，但CysB(T149P)的转录活性会因诱导剂而增加。在2 mM MgCl2条件下，两种蛋白在没有诱导剂时都具有完全活性。第三种突变蛋白CysB(W166R)在10 mM MgCl2时完全无活性，但在2 mM MgCl2时能组成型表达cysK和cysJIH启动子。令人惊讶的是，野生型CysB在2 mM MgCl2时对cysK启动子也是组成型的，但在10 mM MgCl2时不是；在两种浓度下，它激活cysJIH启动子都需要诱导剂。诱变研究表明，启动子之间的这种差异是由于激活位点半位点之间的距离造成的，在cysJIH启动子中它们被1个碱基对隔开，在cysK启动子中被2个碱基对隔开。我们推测，诱导剂的作用是缩短与激活位点相互作用的两个CysB亚基结合结构域之间的距离。野生型和突变型CysB蛋白的体外活性在2 mM MgCl2时与体内行为的相关性比在10 mM MgCl2时好得多，这表明前者是更接近生理状态的浓度。
