Vashee S, Xu H, Johnston S A, Kodadek T
Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
J Biol Chem. 1993 Nov 25;268(33):24699-706.
The GAL4 protein of Saccharomyces cerevisiae is the prototype of a family of transcription factors that contain a "Zn2Cys6" coordination complex in the DNA-binding domain. GAL4 activates the transcription of genes involved in galactose and melibiose metabolism by binding to sites that contain one or more copies of a sequence 5'-CGGN5TN5CCG-3'. Other Zn2Cys6 proteins in S. cerevisiae also recognize sequences containing two CGG triplets, but with different spacings between them. In this report we investigate the mechanism by which GAL4 distinguishes its bona fide binding site from similar sequences as well as from bulk genomic DNA. In vitro, GAL4 recognizes with moderate to high affinity a variety of sites of the general formula (A/C)GGN10-12CCG. This level of specificity is apparently insufficient for the activator to carry out its biological role. However, many of the sites to which GAL4 binds in vitro do not support GAL4-activated transcription in vivo. In most cases there is not a quantitative correlation between the relative affinity of a site for GAL4 in vitro and the level of GAL4-dependent transcription supported by it in vivo. These data imply that there is some mechanism in vivo by which the intrinsic binding specificity of GAL4 is modified.
酿酒酵母的GAL4蛋白是转录因子家族的原型，其DNA结合结构域中含有一个“Zn2Cys6”配位复合物。GAL4通过与包含一个或多个5'-CGGN5TN5CCG-3'序列拷贝的位点结合，激活参与半乳糖和蜜二糖代谢的基因转录。酿酒酵母中的其他Zn2Cys6蛋白也识别含有两个CGG三联体的序列，但它们之间的间距不同。在本报告中，我们研究了GAL4将其真正的结合位点与相似序列以及大量基因组DNA区分开来的机制。在体外，GAL4以中等到高亲和力识别各种通式为(A/C)GGN10-12CCG的位点。这种特异性水平显然不足以使激活剂发挥其生物学作用。然而，GAL4在体外结合的许多位点在体内并不支持GAL4激活的转录。在大多数情况下，一个位点在体外对GAL4的相对亲和力与其在体内支持的GAL4依赖性转录水平之间不存在定量相关性。这些数据表明，体内存在某种机制可改变GAL4的固有结合特异性。
