Jourdan A D, Stauffer G V
Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242, USA.
J Bacteriol. 1998 Sep;180(18):4865-71. doi: 10.1128/JB.180.18.4865-4871.1998.
The GcvA protein is required for both glycine-mediated activation and purine-mediated repression of the gcvTHP operon. Random and site-directed PCR mutagenesis was used to create nucleotide changes in gcvA to identify residues of the protein involved in activation, repression, and DNA binding. Single amino acid substitutions at L30 and F31 cause a defect in activation of a gcvT-lacZ fusion but have no effect on repression or DNA binding. Single amino acid substitutions at V32 and S38 cause the loss of binding of GcvA to DNA. A deletion of the carboxy-terminal 14 amino acids of GcvA results in the loss of purine-mediated repression and, consequently, a constitutive activation of a gcvT-lacZ fusion. The results of this study partially define regions of GcvA involved in activation, repression, and DNA binding and demonstrate that these functions of GcvA are genetically separable.
甘氨酸介导的gcvTHP操纵子激活和嘌呤介导的抑制都需要GcvA蛋白。利用随机和定点PCR诱变在gcvA中产生核苷酸变化,以鉴定该蛋白中参与激活、抑制和DNA结合的残基。L30和F31处的单氨基酸取代导致gcvT-lacZ融合激活缺陷,但对抑制或DNA结合无影响。V32和S38处的单氨基酸取代导致GcvA与DNA结合丧失。GcvA羧基末端14个氨基酸的缺失导致嘌呤介导的抑制丧失,从而导致gcvT-lacZ融合的组成型激活。本研究结果部分定义了GcvA中参与激活、抑制和DNA结合的区域,并证明GcvA的这些功能在遗传上是可分离的。