STRUCTURAL STUDIES OF EUKARYOTIC REPRESSORS

真核基因相关蛋白质的物理相互作用 将使用核磁共振(核磁共振)来研究表达 技巧。酵母中的两种蛋白质，α2和α1 酿酒酵母共同作用结合DNA操纵子并抑制 单倍体特异基因的转录。这两种蛋白质都含有同源异型 结构域，在许多真核转录中发现的DNA结合基序 监管者。这两种蛋白质来自与DNA的三元复合体 包含单倍体特有的操纵子序列 基因。这种三元络合物分别含有α1和α1的一个单体 除操作员DNA外，还有α2。在没有字母2的情况下， α1蛋白对操纵子DNA的亲和力很差。 Alpha2(Alpha128-210)和Alpha1(Alpha166-126)片段，包含 同源域在溶液中形成异二聚体复合体。这种杂二聚体 保持较高的操作员亲和力(Kd=-10-10M)，表明 同源域之间的协同作用。 利用核磁共振方法，我们已经证明了α2的C末端 片段改变了游离蛋白质中无序状态的结构 与异源二聚体中与同源结构域相互作用的螺旋 字母1。将对蛋白质片段进行研究，以确定它们的 溶液结构，以确定分子间的接触 杂二聚体，并表征该络合物与其结合的能力 特定的DNA识别序列。同位素标记方法将是 用于简化对核磁共振谱的解释，并用于鉴定 蛋白质-蛋白质接触。这些研究将提供一些第一批 详细的三维信息，关于 不同的蛋白质以组合的方式相互作用来调节基因 在高等生物中的表达。

The physical interactions of proteins involved in eukaryotic gene expression will be studied using nuclear magnetic resonance (NMR) techniques. Two proteins, alpha2, and alpha1, from the yeast Saccharomyces cerevisiae, act together to bind DNA operators and repress transcription of haploid specific genes. Both proteins contain homeo domains, DNA-binding motifs found in many eukaryotic transcriptional regulators. These two proteins from a ternary complex with DNA containing the operator sequences characteristic of haploid specific genes. This ternary complex contains one monomer each of alpha1 and alpha2 in addition to the operator DNA. In the absence of alpha2, the alpha1 protein has very poor if any affinity for the operator DNA. Fragment of alpha2 (alpha128-210) and alpha1 (alpha166-126), containing the homeodomains form heterodimeric complex in solution. This hetrodimer retains high operator affinity (Kd=-10-10M), suggesting significant cooperative interactions between the homeodomains. Using NMR methods we have demonstrated that the C-terminus of the alpha2 fragment changes structure from a disordered state in the free protein to a helix in the heterodimer that interacts with the homeodomain of alpha1. The protein fragments will be studied to determine their solution structures, to identify the intermolecular contacts of the heterodimer, and to characterize the ability of the complex to bind its specific DNA recognition sequence. Isotope labeling approaches will be used to simplify the interpretation of the NMR- spectra and to identify protein-protein contacts. These studies will provide some of the first detailed three dimensional information concerning the way in which different proteins interact in a combination fashion to regulate gene expression in higher organisms.