Kullik I, Toledano M B, Tartaglia L A, Storz G
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
J Bacteriol. 1995 Mar;177(5):1275-84. doi: 10.1128/jb.177.5.1275-1284.1995.
OxyR is a redox-sensitive transcriptional regulator of the LysR family which activates the expression of genes important for the defense against hydrogen peroxide in Escherichia coli and Samonella typhimurium. OxyR is sensitive to oxidation and reduction, and only oxidized OxyR is able to activate transcription of its target genes. Using site-directed mutagenesis, we found that one cysteine residue (C-199) is critical for the redox sensitivity of OxyR, and a C-199-->S mutation appears to lock the OxyR protein in the reduced form. We also used a random mutagenesis approach to isolate eight constitutively active mutants. All of the mutations are located in the C-terminal half of the protein, and four of the mutations map near the critical C-199 residue. In vivo as well as in vitro transcription experiments showed that the constitutive mutant proteins were able to activate transcription under both oxidizing and reducing conditions, and DNase I footprints showed that this activation is due to the ability of the mutant proteins to induce cooperative binding of RNA polymerase. Unexpectedly, RNA polymerase was also found to reciprocally affect OxyR binding.
OxyR是LysR家族中一种对氧化还原敏感的转录调节因子，它能激活大肠杆菌和鼠伤寒沙门氏菌中对抵御过氧化氢至关重要的基因的表达。OxyR对氧化和还原敏感，只有氧化型的OxyR能够激活其靶基因的转录。通过定点诱变，我们发现一个半胱氨酸残基（C-199）对OxyR的氧化还原敏感性至关重要，C-199→S突变似乎使OxyR蛋白锁定在还原形式。我们还采用随机诱变方法分离出八个组成型活性突变体。所有突变都位于该蛋白的C端区域，其中四个突变位于关键的C-199残基附近。体内和体外转录实验表明，组成型突变蛋白在氧化和还原条件下均能激活转录，DNase I足迹实验表明这种激活是由于突变蛋白诱导RNA聚合酶协同结合的能力。出乎意料的是，还发现RNA聚合酶会反过来影响OxyR的结合。
