Pu W T, Struhl K
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Mol Cell Biol. 1991 Oct;11(10):4918-26. doi: 10.1128/mcb.11.10.4918-4926.1991.
Yeast GCN4 and the Jun oncoprotein are transcriptional activators that bind DNA via a bZIP domain consisting of a leucine zipper dimerization element and an adjacent basic region that directly contacts DNA. Two highly conserved alanines (Ala-238 and Ala-239 in GCN4) and an invariant asparagine (Asn-235) in the basic region have been proposed to play important roles in DNA sequence recognition by bZIP proteins. Surprisingly, these conserved residues can be functionally replaced in GCN4 and in a derivative containing the Jun basic region (Jun-GCN4). The ability of an amino acid to functionally substitute for Asn-235 does not correlate with its preference for assuming the N-cap position of an alpha helix. This finding argues against the proposal of the scissors grip model that the invariant asparagine forms an N cap that permits the basic region to bend sharply and wrap around the DNA. In contrast to a prediction of the induced fork model, the pattern of functional substitutions of the conserved alanines together with the results of uracil interference experiments suggests that Ala-238 and Ala-239 do not make base-specific DNA contacts. Finally, the Jun-GCN4 chimeric proteins appear much more active in vivo than expected from their DNA-binding properties in vitro. The mechanistic and evolutionary implications of these results are discussed.
酵母GCN4和Jun癌蛋白是转录激活因子，它们通过一个bZIP结构域与DNA结合，该结构域由一个亮氨酸拉链二聚化元件和一个直接与DNA接触的相邻碱性区域组成。有人提出，碱性区域中的两个高度保守的丙氨酸（GCN4中的Ala-238和Ala-239）和一个不变的天冬酰胺（Asn-235）在bZIP蛋白识别DNA序列中起重要作用。令人惊讶的是，这些保守残基在GCN4和含有Jun碱性区域的衍生物（Jun-GCN4）中可以被功能性替代。一个氨基酸在功能上替代Asn-235的能力与其占据α螺旋N帽位置的偏好性无关。这一发现与剪刀夹模型的观点相悖，该模型认为不变的天冬酰胺形成一个N帽，使碱性区域能够急剧弯曲并缠绕在DNA上。与诱导叉模型的预测相反，保守丙氨酸的功能替代模式以及尿嘧啶干扰实验的结果表明，Ala-238和Ala-239并不与DNA进行碱基特异性接触。最后，Jun-GCN4嵌合蛋白在体内的活性似乎比根据其体外DNA结合特性所预期的要高得多。本文讨论了这些结果的机制和进化意义。
