Zhou Y N, Chatterjee S, Roy S, Adhya S
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Mol Biol. 1995 Oct 27;253(3):414-25. doi: 10.1006/jmbi.1995.0563.
We isolated and characterized mutant repressors (GalR) of the gal operon in Escherichia coli. These repressors (super-repressors), called GalRs, have a non-inducible phenotype. Repression of the gal operon by super-repressors cannot be lifted by inducer. The mutant galR genes, galRs, have been cloned and the mutational changes determined. Two of them, galRuv7s and galR78s, were located in the proposed sugar binding domains of the repressor. The repressor from wild-type (galR+), as well as from mutant galRuv7s, was purified and characterized biochemically. The results showed that, like wild-type GalR+, GalRuv7s binds to DNA normally and represses transcription from the P1 promoter and stimulates that from the P2 promoter of the gal operon. Nevertheless, compared to GalR+, GalRuv7s is much less sensitive to the presence of the inducer, D-galactose. The affinity of D-galactose to GalRuv7s is 10 to 30-fold lower, as measured by the effect of the inducer on GalR tryptophan fluorescence; GalR complexes with DNA and on GalR repression of transcription. Our results suggest that the super-repressor phenotype of GalRuv7s is because of a defect in D-galactose binding rather than a defect in the ligand-induced allosteric change or increased affinity for the operator.
我们在大肠杆菌中分离并鉴定了半乳糖操纵子的突变阻遏物（GalR）。这些阻遏物（超级阻遏物），即GalRs，具有不可诱导的表型。超级阻遏物对半乳糖操纵子的阻遏不能被诱导剂解除。突变的galR基因，即galRs，已被克隆并确定了突变变化。其中两个，galRuv7s和galR78s，位于阻遏物的推测糖结合结构域中。对野生型（galR+）以及突变型galRuv7s的阻遏物进行了纯化，并进行了生化特性分析。结果表明，与野生型GalR+一样，GalRuv7s能正常结合DNA，阻遏P1启动子的转录，并刺激半乳糖操纵子P2启动子的转录。然而，与GalR+相比，GalRuv7s对诱导剂D-半乳糖的存在不太敏感。通过诱导剂对GalR色氨酸荧光的影响、GalR与DNA的复合物以及GalR对转录的阻遏作用来衡量，D-半乳糖与GalRuv7s的亲和力低10至30倍。我们的结果表明，GalRuv7s的超级阻遏物表型是由于D-半乳糖结合缺陷，而不是配体诱导的变构变化缺陷或对操纵基因的亲和力增加。
