Hieronim Jakubowski MCB 9724929 Editing of Homocysteine by Amino-tRNA Synthetases 1. Technical The non-protein amino acid homocysteine (Hcy), an obligatory precursor of methionine in all cells, poses an accuracy problem for the protein biosynthetic apparatus. Hcy is misactivated by methionyl- (MetRS), isoleucyl- (IleRS), and leucyl-tRNA synthetases (LeuRS) at a frequency exceeding the frequency of translational errors. Misincorporation of Hcy into tRNA and cellular protein is prevented by an editing mechanism of these synthetases which yields a cyclic thioester, Hcy thiolactone. In spite of considerable progress on the elucidation of structures of AARSs, knowledge of the structural basis of editing is limited. This study is undertaken to determine the molecular basis of an error-correcting mechanism, using E. coli class I MetRS as a model, testing a hypothesis that the Zn-binding domain of MetRS constitutes a thiol-binding editing subsite involved in the editing of Hcy. Mutant selection as well as PCR and site-directed mutagenesis methods are used to obtain editing-defective MetRS enzymes. These approaches, combined with sensitive biochemical assays, are designed to allow the study of structure-function relationships both in vitro and in vivo, as well as mapping of the editing subsite and determining its interplay with the synthetic subsite of MetRS. The data will be interpreted within the framework of the three-dimensional structure of MetRS using molecular modeling. This study contributes to the elucidation of the extraordinary accuracy in the flow of genetic information. 2. Non-technical Living organisms have evolved editing or proofreading mechanisms that assure accurate synthesis of their nucleic acids and proteins according to the genetic information contained in DNA. One such mechanism eliminates incorrect amino acids and assures that correct amino acids are incorporated at specific positions of protein chains which is important for proper functioning of these molecules. A parad igm of error correcting mechanisms in protein synthesis is editing of the nonprotein amino acid homocysteine which is being studied at the molecular and cellular levels. This study addresses the molecular details of homocysteine editing, which is fundamental to the extraordinary accuracy in the flow of genetic information.