PROTEIN-NUCLEIC ACID INTERACTIONS

We propose to investigate the interaction between E. coli methionyl-tRNA synthetase (MetRS) and the methionine tRNAs recognized by this enzyme for aminoacylation. We will crosslink tRNAMet to the tRNA binding site of the enzyme and sequence the crosslinked peptides. Protein affinity labeling groups of different length and amino acid specificity will be coupled to specific sites in the tRNA for these crosslinking studies. The results will indicate a number of nucleotide sequences and amino acid sequences which are in close proximity in the protein-nucleic acid complex. This data will allow orientation of different regions of tRNAMet within the three dimensionlal crystal structure of MetRS. In addition, the crosslinking data may assist in defining the conformational changes which occur during binding of the enzyme to cognate tRNAs in solution. The topology of both noncovalent and covalent complexes of tRNAfMet and MetRS will be examined by chemical modification methods. Such studies are expected to provide further details of the dynamic interaction for comparison with models derived from the crystal structure of each component and the peptide sequencing data. Studies on the topology of covalent complexes generated by crosslinking the enzyme to different sites in the tRNA may reveal different steps in the pathway from initial nonspecific tRNA binding to highly specific cognate tRNA recognition. Comparison of the topology of complexes derived from the biologically active monomeric form of MetRS with the native Alpha2 enzyme may help to clarify the structural basis for the anticooperative tRNA binding behavior of the native enzyme. Methionine tRNAs carrying cleavable crosslinking groups will be used to modify specific amino acid residues in the protein for enzyme structure function studies. Identification of a number of essential amino acid residues in the protein is expected to be useful in directing attention to specific regions of the structure for future manipulation by in vitro mutagenesis of the gene, now that its sequence has been established.