PHYSICAL AND GENETICS STUDIES OF REGULATORY PROTEINS

描述：遗传调节是所有生物生存所必需的。 有机体 蛋白质与特定靶DNA序列的结合是 在原核生物和哺乳动物中控制转录起始的机制 真核生物 蛋白质-DNA识别反过来又受到 与其他分子的结合伙伴关系，包括小分子 代谢物以及同聚和异聚蛋白质相互作用。 的 原型原核负转录调节子是lac 阻遏物，一种阻止mRNA起始的同源四聚体蛋白 编码乳糖代谢酶，除非底物在 环境保护 这种蛋白质是一个更大的家族成员， 大肠杆菌中参与转录调控的蛋白质 杆菌 超双胸（UBX）蛋白是同源异型基因的一个成员 一个家族，在广泛的物种中保守，其中每个成员 含有一个DNA识别基序，称为同源结构域。 UBX蛋白 在发育过程中， 果蝇通过改变特定基因的转录。 差异 通过UBX蛋白的调节（激活与抑制）可以在 至少部分来自于与其它植物的同向性和异向性联系 proteins. 本项目的目标是破译 对变构、稳定性、活动调节和组装的贡献 (both同源和异源）。 的 乳糖阻遏物的变构和稳定性机制将被 通过位点特异性诱变进行检查， 基于最近解决的晶体结构的取代 和嘌呤全抑制复合物的结合。 紫胶蛋白N端螺旋-转角-螺旋结构域的结合特性 UBX蛋白的阻遏物和同源结构域通过结合来调节 蛋白质结构的其余部分。 的性质 共价连接的螺旋-转角-螺旋结构域单独和与DNA复合 将被审查。 此外，UBX蛋白中 降低同源结构域对靶DNA位点的亲和力 通过生物化学和遗传学的方法。 区域和特定残基 所需的同源蛋白质-蛋白质组装将在 lac阻遏物和UBX蛋白。 所需的一组残基， 生成一个子单元接口将探索组装的混合 由N-末端螺旋-转角-螺旋DNA结合结构域组成的蛋白质 lac阻遏物，周质糖结合蛋白（通常是单体）， 和二聚体-二聚体所需的阻遏物的C-末端结构域 协会 异源蛋白质-蛋白质结构决定簇 协会将探讨在这些调控蛋白，和合作伙伴 UBX蛋白质将被识别。 这些产生的信息 实验将大大扩展我们对结构的理解 和这些必需的遗传调节蛋白的功能。 因为 与其各自蛋白质的其他成员具有结构同源性 家族，lac阻遏蛋白和UBX蛋白的结果将提供 设计调节蛋白的信息，并将产生一般的 对蛋白质结构和功能原理的深入了解。

DESCRIPTION: Genetic regulation is essential to survival in all living organisms. Protein binding to specific target DNA sequences is a primary mechanism for control of transcription initiation in both prokaryotic and eukaryotic organisms. Protein-DNA recognition in turn is modulated by binding partnerships with other molecules, including both small metabolites and homo- and heteromeric protein interactions. The prototypic prokaryotic negative transcriptional regulator is lac repressor, a homotetrameric protein that prevents initiation of mRNA encoding the lactose metabolic enzymes unless substrate is available in the environment. This protein is a member of a larger family of proteins that participate in transcriptional regulation in Escherichia coli. The Ultrabithorax (UBX) protein is a member of the homeotic gene family, conserved among a broad range of species, in which each member contains a DNA recognition motif known as the homeodomain. UBX protein is involved in specifying segmental identity during development in Drosophila by altering transcription of specific genes. Differences in regulation by UBX protein (activation vs. repression) may be derived at least in part from homotropic and heterotropic associations with other proteins. The goal of this project period is to decipher structural contributions to allostery, stability, activity modulation, and assembly (both homo- and heteromeric) in these two regulatory proteins. The mechanisms of allostery and stability in the lac repressor will be examined by site-specific mutagenesis with residues selected for substitution based on the recently solved crystallographic structures of the core domain of this protein and the purine holorepressor complex. Binding properties of the N-terminal helix-turn-helix domain of lac repressor and homeodomain of UBX protein are modulated by association with the remainder of the protein structure. The properties of covalently linked helix-turn-helix domains alone and complexed with DNA will be examined. In addition, the regions of the UBX protein that diminish homeodomain affinity for target DNA sites will be identified by biochemical and genetic methods. Regions and specific residues required for homologous protein-protein assembly will be examined in both lac repressor and UBX protein. The set of residues necessary to generate a subunit interface will be explored by assembly of a hybrid protein consisting of the N-terminal helix-turn-helix DNA binding domain of lac repressor, periplasmic sugar binding protein (normally monomeric), and the C-terminal domain of repressor required for dimer-dimer association. Structural determinants for heterologous protein-protein association will be explored in these regulatory proteins, and partners for UBX protein will be identified. The information generated by these experiments will expand significantly our understanding of the structure and function of these essential genetic regulatory proteins. Because of structural homology with other members of their respective protein families, the results on lac repressor and UBX proteins will provide information for designing regulatory proteins and will yield general insights into principles of protein structure and function.