The objective of this proposal is to elucidate how a class II aminoacyl-tRNA synthetase specifically recognizes its cognate RNA substrates discriminates against non-cognate tRNAs and discriminates against non-cognate tRNAs. The studies will be carried out with Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) whose gene is cloned and sequenced, and which can be functionally expressed both in yeast and in Escherichia coli. This gives us the great advantage of using abacterial host for selected experiments. The availability of crystal structure of the E. coli SerRS provides the basis for the interesting comparative study.
The specific aims of the project are: 1) Identification of tRNA-binding domains in the SerRS protein by performing extensive in vitro mutagenesis of the serS gene. The mutants will be analyzed in yeast and bacteria. The facile method for overproduction and purification of yeast SerRS from E. coli will be employed for preparation of the mutant enzymes for biochemical analysis in vitro. 2) Construction of E. coli-like yeast SerRS, by deletion of the region encoding the C-terminal extension if the eukaryotic protein. 3) The recognition of heterologous cognate and non-cognate tRNAs by SerRS. 4) Determination of yeast tRNA(Ser) identity elements by using already available set of mutants of Schizosaccharomyces pombe suppressor tRNA(Ser). 5) Cloning the gene for S. pombe SerRS by complementation of S. cerevisiae null allele strain. These studies should complement and extend the ongoing project on tRNA recognition by class I synthetases, involving mainly E. coli tRNA(Gln): GlnRS system.
