NSF Award
9018979
Aminoacyl-tRNA synthetases esterify amino acids to cognate tRNAS during the course of protein synthesis and are thus essential to the decoding of genetic information in all organisms. The objectives of the work proposed here are to determine the role of zinc in the aminoacylation and/or DNA binding activities of E. coli alanyl-tRNA synthetase (ARS) and to elucidate structural elements that coordinate with this metal. The function of zinc in ARS will be explored through a series of denaturation, kinetic, and DNA binding experiments, and the location of the metal binding site will be determined through a zinc-blotting procedure and atomic absorption spectroscopy. Furthermore, the hypothesis that this enzyme contains a metal binding domain resembling a zinc finger will be tested by chemical modification and site-directed mutagenesis. Such a domain may be involved in the autoregulation of ARS synthesis, which is thought to be mediated through ARS binding to the promoter region of its gene. Alternatively, the metal may be involved in the protein-RNA interaction, as is the case in retroviral zinc finger proteins. The demonstration of either case would be novel for this important class of enzymes.
