TRNA RECOGNITION BY E COLI AMINOACYL TRNA SYNTHETASES

氨基酸与转运RNA（tRNA）的正确连接是 这是翻译忠实度的主要决定因素。氨酰-tRNA 合成酶（AAS）通过正确地调节细胞的功能来维持这种关键的细胞功能。 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 导致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 因此，在非同源tRNA中一定存在负元件 这会阻碍错酰化。积极和消极的元素可以是 tRNA或单个核苷酸的结构特征。 该项目研究了tRNAs中的元件类型，这些元件解释了 保持氨基酸的特异性。 构建了一系列tRNASer和tRNALeu突变体并进行了分析 从两个方面。体外氨酰化动力学将用于定义 对于给定的AAS，底物的相对效率。体内 抑制系统将被用来定义其在小区中的身份。从 通过比较这些体外和体内结果，我们将确定 积极和消极的因素。我们也会用结果做检验 底物的体外最佳性是否与 它在体内的身份和正负平衡 元件是维持细胞中氨基酸特异性所必需的。 我们对受体茎突变体的体外动力学分析表明，SerAAS 识别受体茎核苷酸的方式出乎意料的 其他工人的理论分析。通过使用核苷酸类似物， 将调查SerAAS与各职能组之间的联系 螺旋状RNA我们还计划进行一项全面调查，以确定 AAS以相似或不同的方式区分螺旋RNA。的 这些研究结果不仅适用于tRNA/AAS 相互作用，也涉及许多生物系统， RNA被蛋白质识别。 我们的体内和体外研究表明，tRNASer的额外茎/环 也是SerAAS的一个重要的积极认可因素。我们将 研究所需的额外茎/环的结构和序列， 使用Turk和Gold的SELEX程序通过SerAAS识别。的 额外茎/环结合的相互依赖性和 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， 茎/环和受体茎被改变。

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.