Suckow M, Madan A, Kisters-Woike B, von Wilcken-Bergmann B, Müller-Hill B
Institut für Genetik, Universität zu Köln, Germany.
Nucleic Acids Res. 1994 Jun 25;22(12):2198-208. doi: 10.1093/nar/22.12.2198.
The specificity of the GCN4/DNA complex is mediated by a complicated network of interactions between the basic regions of both GCN4 monomers and their target halfsites. According to X-ray analyses (1, 2) one particular thymine of the target sequence is recognized by serine -11 and alanine -15 (we define the leucine in the first d-position of the heptad repeats as +1). We replaced serine -11 or alanine -15 with all other amino acids and analysed the DNA binding properties of the resulting stable GCN4 derivatives by electrophoretic mobility shift assays. Among these, mutants with tryptophan in position -11, or glutamic acid and glutamine in position -15, differ significantly from GCN4 in their DNA binding specificities. We then constructed selected double mutants, which differ from GCN4 in positions -11, -15 or -14 (3) of the basic region. The double mutants with tryptophan in position -11 and asparagine or serine in position -14 show drastically altered DNA binding specificities, presumably due to additive effects.
GCN4/DNA复合物的特异性是由GCN4两个单体的碱性区域与其靶半位点之间复杂的相互作用网络介导的。根据X射线分析（1,2），靶序列的一个特定胸腺嘧啶由丝氨酸-11和丙氨酸-15识别（我们将七肽重复序列第一个d位置的亮氨酸定义为+1）。我们将丝氨酸-11或丙氨酸-15替换为所有其他氨基酸，并通过电泳迁移率变动分析来分析所得稳定GCN4衍生物的DNA结合特性。其中，-11位为色氨酸或-15位为谷氨酸和谷氨酰胺的突变体，其DNA结合特异性与GCN4有显著差异。然后我们构建了选定的双突变体，它们在碱性区域的-11、-15或-14位（3）与GCN4不同。-11位为色氨酸且-14位为天冬酰胺或丝氨酸的双突变体显示出DNA结合特异性发生了剧烈变化, 这可能是由于累加效应。
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