GCN4, A EUKARYOTIC TRANSCRIPTIONAL ACTIVATOR PROTEIN, BINDS AS A DIMER TO TARGET DNA
GCN4, A EUKARYOTIC TRANSCRIPTIONAL ACTIVATOR PROTEIN, BINDS AS A DIMER TO TARGET DNA
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DOI：
10.1002/j.1460-2075.1987.tb02573.x
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发表时间：
1987-09-01
期刊：
影响因子：
11.4
通讯作者：
STRUHL, K
中科院分区：
文献类型：
--
作者：
HOPE, IA;STRUHL, K
从克隆的基因体外合成的真核转录激活蛋白Gcn4与酵母氨基酸生物合成基因的启动子特异结合。先前对截短的GCN4衍生物的分析将DNA结合域定位于C-末端60个氨基酸，并发现GCN4衍生物的大小与蛋白质-DNA复合体的凝胶迁移率呈负相关。这一观察结果被用来发展一种新的方法来确定DNA结合蛋白的亚基结构。野生型Gcn4蛋白和较小的Gcn4衍生物的混合物产生了三个与DNA的复合体，其中两个对应于蛋白质单独存在时观察到的复合体，以及一个新的中等迁移率的复合体。这种额外的复合体是由两种不同大小的Gcn4蛋白的异源二聚体引起的，表明Gcn4以二聚体的形式与DNA结合。足以进行二聚化的接触定位于60C端氨基酸的DNA结合域，这表明GCN4的二聚化是特异性DNA结合的关键方面。此外，在没有靶DNA的情况下，可以形成稳定的Gcn4二聚体。这些观察结果提出了一种Gcn4蛋白的结构模型，在该模型中，二聚体结合到重叠的和不相同的半位点，解释了为什么Gcn4识别位点在刺激转录方面起双向作用。
The eukaryotic transcriptional activator protein, GCN4, synthesized in vitro from the cloned gene, binds specifically to the promoters of yeast amino acid biosynthetic genes. Previous analysis of truncated GCN4 derivatives localized the DNA binding domain to the C-terminal 60 amino acids and revealed that the size of the GCN4 derivative and the electrophoretic mobility of the protein-DNA complex were inversely related. This observation was utilized here to develop a novel method for determining the subunit structure of DNA binding proteins. A mixture of wild-type GCN4 protein and a smaller GCN4 derivative generated three complexes with DNA, two corresponding to those observed when the proteins are present individually and one new complex of intermediate mobility. This extra complex results from the heterodimer of the two GCN4 proteins of different sizes, demonstrating that GCN4 binds DNA as a dimer. The contacts sufficient for dimerization were localized to the 60 C-terminal amino acid, DNA binding domain, suggesting that dimerization of GCN4 is a critical aspect of specific DNA binding. Furthermore, stable GCN4 dimers were formed in the absence of target DNA. These observations suggest a structural model of GCN4 protein in which a dimer binds binds to overlapping and nonidentical half-sites, explaining why GCN4 recognition sites act bidirectionally in stimulating transcription.