Bates D M, Lazazzera B A, Kiley P J
Department of Bacteriology, University of Wisconsin, Madison 53706, USA.
J Bacteriol. 1995 Jul;177(14):3972-8. doi: 10.1128/jb.177.14.3972-3978.1995.
In order to gain insight into the mechanism by which the Escherichia coli transcription factor FNR* is activated in response to anaerobiosis, we have analyzed FNR mutant proteins which, unlike the wild-type protein, stimulate gene expression in the presence of oxygen in vivo. Cell extracts containing seven different FNR* mutant proteins were tested in vitro for the ability to bind to the FNR consensus DNA site in a gel retardation assay under aerobic conditions. At the concentration of protein tested, only extracts which contained FNR* mutant proteins with amino acid substitutions at position 154 showed significant DNA binding. The three position-154 FNR* mutant proteins could be further distinguished from the other mutant proteins by analysis of the in vivo phenotypes of FNR* proteins containing amino acid substitutions at either of two essential cysteine residues. In the presence of oxygen, FNR* mutant proteins with amino acid substitutions at position 154 were the least affected when either Cys-23 or Cys-122 was substituted for Ser. On the basis of these in vivo and in vitro analyses, FNR* mutant proteins appear to segregate into at least two classes. Thus, it appears that each class of FNR* substitutions alters the normal pathway of FNR activation in response to oxygen deprivation by a different mechanism.
为了深入了解大肠杆菌转录因子FNR在厌氧条件下被激活的机制，我们分析了FNR突变蛋白，这些突变蛋白与野生型蛋白不同，在体内有氧存在时能刺激基因表达。在需氧条件下，通过凝胶阻滞试验，对含有七种不同FNR突变蛋白的细胞提取物进行体外测试，以检测其与FNR共有DNA位点结合的能力。在所测试的蛋白质浓度下，只有含有在第154位氨基酸发生取代的FNR突变蛋白的提取物显示出显著的DNA结合。通过分析在两个必需半胱氨酸残基之一处含有氨基酸取代的FNR蛋白的体内表型，可将三种第154位的FNR突变蛋白与其他突变蛋白进一步区分开来。在有氧存在时，当Cys-23或Cys-122被丝氨酸取代时，第154位氨基酸发生取代的FNR突变蛋白受影响最小。基于这些体内和体外分析，FNR突变蛋白似乎至少可分为两类。因此，似乎每一类FNR取代通过不同机制改变了FNR在缺氧时的正常激活途径。
