SUBUNIT FUNCTION AND TOPOLOGY IN GLYCYL-TRNA SYNTHETASE

本研究探讨了这两种类型的功能和组织， 包含甘氨酰-tRNA合成酶（α 2 β 2）的亚基。 在这类酶中，不同亚基的存在是罕见的， 目前的工作是为了了解如何和为什么他们被用于 酶的生物学作用，转移RNA的氨酰化，以及在一个 提出了新的作用，其结构基因glyS的调控。 具体而言，电子显微镜和电泳研究 酶的交联样品将用于改进我们的模型， 酶的整体结构和亚基拓扑结构。 这些数据将提供 为理解和综合关于其职能的数据奠定坚实基础 亚单位。 位点特异性修饰试剂将用于化学修饰。 标记酶上的ATP结合位点（甘氨酸活化位点）和 tRNA的3 ′末端（甘氨酸酯化位点）以及 与tRNA分子的受体茎连接。 这些研究 将允许定位和定量的网站上的一个或另一个 亚单位的类型。 另外，从所述肽中分离标记的肽是可行的。 修饰的酶将使我们开始绘制功能域，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用于探测 这些网站，并询问实验是否相关的酶， 结构上与甘氨酰-tRNA合成酶同源。 最后，直接绑定 和使用小（小于900 bp）DNA的核酸酶保护实验 含有glyS启动子区和纯化的甘氨酰-tRNA的片段 合成酶将探索相互作用的强度和特异性， glyS通过其基因产物自我调节的可能性。 丙氨酰-tRNA合成酶和alaS DNA的平行研究， 自动调节系统，将完成比较，以更好地 描述alaS法规的特征。 总之，这些研究构成了第一个系统研究， 一种罕见α 2 β 2）氨酰-tRNA合成酶亚基功能。 这些 研究应大大增加我们对机制的了解， 调节蛋白质生物合成的基本和必要反应。

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.