Saviola B, Seabold R R, Schleif R F
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
J Bacteriol. 1998 Aug;180(16):4227-32. doi: 10.1128/JB.180.16.4227-4232.1998.
We sought a mutation in the DNA binding domain of the arabinose operon regulatory protein, AraC, of Escherichia coli that allows the protein to bind DNA normally but not activate transcription. The mutation was isolated by mutagenizing a plasmid overproducing a chimeric leucine zipper-AraC DNA binding domain and screening for proteins that were trans dominant negative with regard to wild-type AraC protein. The mutant with the lowest transcription activation of the araBAD promoter was studied further. It proved to alter a residue that had previously been demonstrated to contact DNA. Because the overproduced mutant protein still bound DNA in vivo, it is deficient in transcription activation for some reason other than absence of DNA binding. Using the phase-sensitive DNA bending assay, we found that wild-type AraC bends DNA about 90 degrees whereas the mutant bends DNA by a smaller amount.
我们在大肠杆菌阿拉伯糖操纵子调节蛋白AraC的DNA结合结构域中寻找一种突变，该突变能使该蛋白正常结合DNA，但不激活转录。通过对过量表达嵌合亮氨酸拉链 - AraC DNA结合结构域的质粒进行诱变，并筛选对野生型AraC蛋白具有反式显性负效应的蛋白质，分离出了该突变。对araBAD启动子转录激活作用最低的突变体进行了进一步研究。结果证明它改变了一个先前已证明与DNA接触的残基。由于过量表达的突变蛋白在体内仍能结合DNA，其转录激活缺陷是由DNA结合缺失以外的某种原因导致的。使用相敏DNA弯曲试验，我们发现野生型AraC使DNA弯曲约90度，而突变体使DNA弯曲的程度较小。
