SUBUNIT FUNCTION AND TOPOLOGY IN GLYCYL-TRNA SYNTHETASE

本研究探讨了这两种类型的语言的功能和组织 组成甘氨酰-tRNA合成酶(α2β2)的亚基。 在这类酶中，不同亚基的存在是罕见的 目前的工作旨在了解它们是如何以及为什么在 该酶的生物学作用，转移RNA的氨酰化，以及在 提出了新的作用，调控其结构基因GlyS。 具体地说，电子显微镜和电泳法研究 这种酶的交联样本将被用来改进我们的模型 酶的整体结构和亚基拓扑结构。这些数据将提供一个 为理解和综合关于智能交通系统功能的数据奠定坚实的基础 亚单位。将使用特定部位的修饰试剂进行化学修饰 酶上的标记结合位点与ATP(甘氨酸激活位点)和 TRNA的3‘末端(甘氨酸酯化位点)以及一个区域 与tRNA分子的受体茎连接。这些研究 将允许对其中之一上的站点进行本地化和量化 子单位的类型。此外，标记多肽的分离也是非常重要的。 经过修饰的酶将使我们能够开始绘制包括 已知氨基酸序列中的核酸识别区域。 亚基特异性抗体也将被用来探测 并通过实验询问相关的酶是否 在结构上与甘氨酰-tRNA合成酶同源。最后，直接绑定 以及使用小分子(小于900bp)DNA进行的核酸酶保护实验 含有GlyS启动子区域的片段和纯化的甘氨酰-tRNA 合成酶将探索相互作用的强度和特异性，并 由其基因产物自动调节糖链的激动人心的可能性。 丙氨酰-tRNA合成酶和已知基因ALAS DNA的平行研究 自动调节系统，将会完善以供比较和完善 描述一下ALAS监管的特点。 综上所述，这些研究构成了第一个系统的 亚基在一种罕见的α-β-tRNA合成酶中起作用。这些 研究应该大大增强我们对这一机制的理解和 调节蛋白质生物合成的一个基本和必要的反应。

This research explores the function and organization of the two types of subunits which comprise the enzyme glycyl-tRNA synthetase (Alpha2 Beta2). The existence of dissimilar subunits is rare in this class of enzymes and the present work is aimed at understanding how and why they are used in the biological role of the enzyme, the aminoacylation of transfer RNA, and in a proposed new role, the regulation of its structural gene, glyS. Specifically, electron microscopic and electrophoretic studies on cross-linked samples of the enzyme will be used to refine our model for the enzyme's overall structure and subunit topology. These data will provide a firm basis for understanding and synthesizing data on the function of its subunits. Site-specific modification reagents will be used to chemically label binding sites on the enzyme for ATP (glycine activation site) and for the 3' terminus of tRNA (glycine esterification site) as well as a region interfacing with the acceptor stem of the tRNA molecule. These studies will allow the localization and quantitation of sites on one or the other type of subunit. Additionally, the isolation of labeled peptides from the modified enzymes will allow us to begin mapping functional domains incuding nucleic acid-recognition regions in the known amino acid sequence. Subunit-specific antibodies will also be employed to probe the location of these sites and to ask experimentally whether related enzymes are structurally homologous to glycyl-tRNA synthetase. Finally, direct binding and nuclease protection experiments using small (less than 900 bp) DNA fragments containing the glyS promoter region and purified glycyl-tRNA synthetase will explore the strength and specificity of the interaction and the exciting possibility of autoregulation of glyS by its gene product. Parallel studies on alanyl-tRNA synthetase and alaS DNA, a known autoregulatory system, will be completed for comparison and to better characterize the alaS regulation. Taken together, these studies constitute the first systematic study of subunit function in a rare Alpha2 Beta2) aminoacyl-TRNA synthetase. These studies should augment greatly our understanding of the mechanism and regulation of a fundamental and essential reaction of protein biosynthesis.