STRUCTURAL BASIS OF RNA-CATALYZED AMINOACYLATION OF RNA

描述(申请人提供)：氨基酸与适配RNA分子的结合是蛋白质生物合成中的一个关键反应。氨基酰化反应在细胞内由氨酰基-tRNA合成酶催化。最近，通过体外筛选获得了三类催化RNA(核酶)，它们通过将氨基酰基部分从预先形成的腺苷转移到自己的3‘-末端来概括这一活性。第一类包含接受任何氨基酰基的RNA，而第二类成员是苯丙氨酸和酪氨酸所特有的。第三类核酶还催化二肽的形成，这一反应类似于核糖体这种大的核酶所催化的反应。在这个方案中，这三类自氨酰化RNA的三维结构将通过X射线结晶学来确定。核酶将在底物和产物类似物的络合物中进行分析。本研究获得的结构信息将揭示这些核酶的活性部位的构型、氨基酸特异性结合口袋的组织结构以及催化机理。这将有助于澄清氨酰基转移到多肽或RNA是否涉及熵催化(即底物的精确定位)，或者它是否需要酶上的特殊催化基团，如酸或碱；因此，它将增加我们对较大生物系统(如氨基酰-tRNA合成酶和核糖体)催化反应的理解。

DESCRIPTION (provided by applicant): Attachment of amino acids to adapter RNA molecules is a key reaction in protein biosynthesis. The aminoacylation reaction is catalyzed in the cell by aminoacyl-tRNA synthetases. Recently, three classes of catalytic RNAs (ribozymes) that recapitulate this activity by transferring the aminoacyl moiety from a pre-formed adenylate to their own 3'-termini have been obtained through in vitro selection. The first class contains RNAs that accept any aminoacyl moiety, whereas members of the second class are specific for phenylalanine and tyrosine. Ribozymes of the third class also catalyze the formation of dipeptides, a reaction analogous to that catalyzed by a large ribozyme, the ribosome. In this proposal, the three-dimensional structures of self-aminoacylating RNAs of the three classes will be determined by x-ray crystallography. The ribozymes will be analyzed in complex with substrate and product analogs. The structural information obtained in this study will reveal the configuration of the active site of these ribozymes, organization of the amino acid-specific binding pocket and the mechanism of catalysis. It will help clarify the issue of whether the aminoacyl transfer to a peptide or an RNA involves entropic catalysis (i.e., precise positioning of the substrates) or it requires special catalytic groups on the enzyme, such as an acid or base; hence, it will add to our understanding of the reactions catalyzed by the larger biological systems such as the aminoacyl-tRNA synthetases and the ribosome.