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.
