STRUCTURAL STUDIES OF EUKARYOTIC REPRESSORS

真核生物基因组中蛋白质间的物理相互作用 表达将使用核磁共振（NMR）进行研究 技术. 酵母中的两种蛋白质，α 2和α 1， 酿酒酵母，共同作用，以结合DNA操纵子和抑制 单倍体特异基因的转录。 这两种蛋白质都含有homeo 结构域，DNA结合基序，发现在许多真核生物转录 监管部门 这两种蛋白质与DNA形成三元复合物 含有单倍体特异性的操纵子序列， 基因. 该三元复合物含有α 1和α 2中的每一种单体。 alpha 2除了操纵基因。 在没有α 2的情况下， α 1蛋白对操纵基因DNA的亲和性非常差。 α 2（α 128 -210）和α 1（α 166 -126）的片段，含有 同源结构域在溶液中形成异二聚体复合物。 这种异二聚体 保留了高的操纵子亲和力（Kd=-10-10M），表明显著的 homeodomains之间的相互作用。 使用NMR方法，我们已经证明α 2的C-末端 片段从游离蛋白质的无序状态改变结构 与异二聚体中的螺旋相互作用， α 1。 将研究蛋白质片段以确定其 溶液结构，以确定分子间的接触， 异二聚体，并表征复合物结合其 特异性DNA识别序列。 同位素标记方法将是 用于简化NMR光谱的解释并识别 蛋白质接触 这些研究将提供一些第一批 详细的三维信息， 不同的蛋白质以组合的方式相互作用， 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.