Mechanisms underlying the specificity of aminoacyl-tRNA synthesis will be studied in a variety of experimental systems. First, recently determined structures of E. coli cysteinyl-tRNA synthetase will be used as a basis for exploring unique mechanisms underlying the selectivities for amino acid and tRNA in that system. A zinc-mediated conformational switch controlling placement of the tRNA 3'-end in the active site will be studied, and the origins of shape-selective tRNA recognition will be explored in complementary studies of human and E. coli CysRS. Next, the mechanistic basis for the coupling of amino acid and tRNA specificities will be studied in E. coli glutaminyl-tRNA synthetase. In this case the primary approach will be the application of newly-developed transient kinetic methods. Results of these experiments will inform the design of amino acid specificity switches by rational mutagenesis, with a view towards introduction of activity towards noncognate and nonstandard amino acids. Finally, the mechanisms by which the H. pylori tRNA- dependent amidotransferase converts misacylated tRNAs into suitable substrates for protein synthesis will be studied. Experiments here will focus on establishing the identities of the tRNA recognition elements and some aspects of the reaction pathway. The elucidation of how induced fit and indirect readout mechanisms control tRNA and amino acid specificities in model tRNA synthetases will be relevant to understanding such processes in more complex particles such as the ribosome. Further, there is great potential for developing novel antimicrobial compounds based both on discrimination between human and bacterial synthetases, and on the properties of bacterial tRNA amidotransferases, which possess no human counterparts. Finally, engineering of tRNA synthetases to expand the genetic code may open the door to novel protein-based therapeutics and has the potential to impact the development of therapies for many human diseases.