Ostrowski J, Kredich N M
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
J Bacteriol. 1989 Jan;171(1):130-40. doi: 10.1128/jb.171.1.130-140.1989.
The cysJIH promoter regions from Salmonella typhimurium LT7 and Escherichia coli B were cloned and sequenced. Primer extension analyses showed that the major in vivo transcription initiation site in S. typhimurium is located 171 nucleotides upstream of the cysJ start codon. Minor start sites were found 8 and 9 nucleotides downstream of the major site. In vivo transcription initiation in E. coli was found to occur at a single site 66 nucleotides upstream of the cysJ start codon. Primer extension studies also indicated that chromosomal cysJIH transcription is stimulated by sulfur limitation and repressed by growth on L-cystine. Paradoxically, in strains carrying plasmids containing the S. typhimurium cysJIH region, the highest levels of primer extension products were found with RNA from cells grown on L-cystine, even though levels of the proteins encoded by cysJ and cysI were normally repressed. In vitro transcription runoff studies with DNA template from the S. typhimurium cysJIH promoter region showed synthesis of a product originating at the major in vivo start site, which was dependent on the presence of purified cysB protein and either O-acetyl-L-serine or N-acetyl-L-serine. N-Acetyl-L-serine was 10- to 30-fold more active than O-acetyl-L-serine as an in vitro inducer of cysJIH transcription.
对鼠伤寒沙门氏菌LT7和大肠杆菌B的cysJIH启动子区域进行了克隆和测序。引物延伸分析表明，鼠伤寒沙门氏菌体内主要的转录起始位点位于cysJ起始密码子上游171个核苷酸处。在主要位点下游8和9个核苷酸处发现了次要起始位点。发现大肠杆菌体内的转录起始发生在cysJ起始密码子上游66个核苷酸处的单个位点。引物延伸研究还表明，染色体cysJIH转录受到硫限制的刺激，并受到L-胱氨酸生长的抑制。矛盾的是，在携带含有鼠伤寒沙门氏菌cysJIH区域质粒的菌株中，即使cysJ和cysI编码的蛋白质水平通常受到抑制，但从在L-胱氨酸上生长的细胞中提取的RNA中发现了最高水平的引物延伸产物。用来自鼠伤寒沙门氏菌cysJIH启动子区域的DNA模板进行的体外转录延伸研究表明，合成的产物起始于体内主要起始位点，这依赖于纯化的cysB蛋白以及O-乙酰-L-丝氨酸或N-乙酰-L-丝氨酸的存在。作为cysJIH转录的体外诱导剂，N-乙酰-L-丝氨酸的活性比O-乙酰-L-丝氨酸高10至30倍。
