Johnston S A, Zavortink M J, Debouck C, Hopper J E
Proc Natl Acad Sci U S A. 1986 Sep;83(17):6553-7. doi: 10.1073/pnas.83.17.6553.
In the yeast Saccharomyces cerevisiae regulation of the galactose/melibiose regulon rests on a dosage-dependent functional interplay between the positive regulator of transcription, the GAL4 protein, and the negative regulator, GAL80 protein. We have used this interplay to select in vitro generated fusions between the yeast ADH1 promoter and GAL4 coding sequence that overproduce GAL4 protein, allowing the identification of GAL4 protein in crude extracts from yeast. One type of these constructions produces a GAL4 protein that lacks its normal NH2 terminus. This protein is unable to complement a gal4 lesion but still retains a domain that functionally antagonizes the negative regulatory protein. One defect in this truncated protein is its inability to be concentrated in the nucleus. However, the nuclear localization defect is complemented by full-length GAL4 protein. The truncated protein also appears to effect changes in the relative transcriptional levels of the structural genes. These observations imply that GAL4 protein consists of several domains, including ones for nuclear localization, interaction with the negative regulatory protein, and, possibly, separable transcriptional activation domains for the structural genes.
在酿酒酵母中，半乳糖/蜜二糖调节子的调控取决于转录正调节因子GAL4蛋白与负调节因子GAL80蛋白之间剂量依赖性的功能相互作用。我们利用这种相互作用，在体外筛选酵母ADH1启动子与GAL4编码序列之间产生的融合体，这些融合体过量产生GAL4蛋白，从而能够在酵母粗提物中鉴定出GAL4蛋白。这些构建体中的一种产生了一种缺少正常NH2末端的GAL4蛋白。这种蛋白不能互补gal4损伤，但仍保留一个在功能上拮抗负调节蛋白的结构域。这种截短蛋白的一个缺陷是它不能集中在细胞核中。然而，全长GAL4蛋白可弥补核定位缺陷。截短蛋白似乎也会影响结构基因相对转录水平的变化。这些观察结果表明，GAL4蛋白由几个结构域组成，包括用于核定位的结构域、与负调节蛋白相互作用的结构域，以及可能用于结构基因的可分离转录激活结构域。
