TRNA RECOGNITION BY E COLI AMINOACYL-TRNA SYNTHETASES

氨基酸与转移RNA(TRNAs)的正确结合是 翻译保真度的主要决定因素。氨酰-tRNA 合成酶(AsS)通过正确地维持这一重要的细胞功能 将它们的同源tRNA与其他19个非同源组区分开来 在牢房里。每个AAS必须识别其同源词中的积极元素 在两者之间产生有效相互作用的tRNA 大分子。然而，这些元件可以存在于非同源tRNA中 也是。因此，非同源tRNAs中必然含有否定成分 这会阻碍错酰化反应。正元素和负元素可以是 不是tRNA的结构特征，就是单个核苷酸。 这个项目调查了tRNA中解释 维持氨基酸的专一性。 将构建一系列tRNASer和tRNALeu突变体并进行检测 在两个方面。体外氨酰化动力学将被用来定义 对于给定的原子吸收光谱，底物的相对效率。一个活体内 将使用抑制系统来定义其在单元格中的身份。从… 将这些体外和体内结果进行比较，我们将确定 积极因素和消极因素。我们也会用成绩来做检查 底物的体外最佳性是否成正比 它在体内的身份以及积极和消极的平衡 这些元素是维持细胞内氨基酸专一性所必需的。 我们对受体茎突变体的体外动力学分析表明，血清原子吸收光谱显示 以意想不到的方式识别受体茎核苷酸 其他工人的理论分析。通过使用核苷酸类似物我们 将调查SerAAS与功能小组之间的联系 螺旋核糖核酸。我们还计划进行一次全面的调查，以确定 AASS以相似或不同的方式区分螺旋RNA。这个 这些研究的结果将不仅适用于tRNA/AAS 相互作用，也涉及到许多涉及 蛋白质对RNA的识别。 我们的体内和体外研究表明，tRNASer的额外茎/环 对SerAAS来说，这也是一个重要的积极认可因素。我们会 研究所需的额外茎/环的结构和序列 使用Turk和Gold的SELEX程序进行SerAAS识别。这个 额外茎/环结合的相互依赖和对 受体茎核苷酸将通过体外动力学分析进行研究 在模型RNA中，额外的 阀杆/环和受体阀杆被改变。

The correct attachment of amino acids to transfer RNAs (tRNAs) is the primary determinant of the fidelity of translation. The aminoacyl-tRNA synthetases (AASs) maintain this critical cellular function by correctly distinguishing their cognate tRNAs from the 19 other noncognate groups in the cell. Each AAS must recognize positive elements in its cognate tRNA that result in a productive interaction between the two macromolecules. However, these elements can exist in non-cognate tRNAs as well. Therefore, there must be negative elements in non-cognate tRNAs that discourage misacylation. Positive and negative elements can be either structural characteristics of the tRNA or individual nucleotides. This project investigates the types of elements in tRNAs that account for the maintenance of amino acid specificity. A series of tRNASer and tRNALeu mutants will he constructed and assayed in two ways. In vitro aminoacylation kinetics will be used to define the relative efficiency of a substrate for a given AAS. An in vivo suppression system will be used to define its identity in the cell. From a comparison of these in vitro and in vivo results, we will determine the positive and negative elements. We will also use the results do examine whether the in vitro optimality of a substrate is directly proportional to its in vivo identity and what balance of positive and negative elements is required to maintain amino acid specificity in the cell. Our in vitro kinetic analyses of acceptor stem mutants showed that SerAAS recognizes the acceptor stem nucleotides in ways unanticipated by the theoretical analyses of other workers. By using nucleotide analogues we will investigate contacts made between SerAAS and the functional groups of helical RNA. We also plan a comprehensive survey to determine whether AASs discriminate among helical RNA in similar or different ways. The results of these studies will be applicable to not only tRNA/AAS interactions but also to the many biological systems that involve the recognition of RNA by proteins. Our in vivo and in vitro studies show that the extra stem/loop of tRNASer is also an important positive recognition element for SerAAS. We will study the structure and sequence of the extra stem/loop required for recognition by SerAAS using the SELEX procedure of Turk and Gold. The interdependence of binding of the extra stem/loop and the recognition of acceptor stem nucleotides will be studied by in vitro kinetic analyses of model RNAs in which the physical connection between the extra stem/loop and acceptor stem are altered.