TRANSFER RNA-DEPENDENT COGNATE AMINO-ACID RECOGNITION BY AN AMINOACYL-TRANSFER-RNA SYNTHETASE

An investigation of the role of tRNA in the catalysis of aminoacylatio n by Escherichia coli glutaminyl-tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNA(Gln) determines amino acid affinity. Mutations in GlnRS at D235, which mak es contacts with nucleotides in the acceptor stem of tRNA(Gln), and at R260 in the enzyme's active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of m utants of GlnRS at position 235, showed amino acid recognition to be t RNA mediated. Aminoacylation of tRNA(CUA)(Tyr) [tyrT (UAG)] by GlnRS-D 235H resulted in a 4-fold increase in the K-m for Gln, which was reduc ed to a 2-fold increase when A73 was replaced with G73. These and prev ious results suggest that specific interactions between GlnRS and tRNA (Gln) ensure the accurate positioning of the 3' terminus. Disruption o f these interactions can change the K-m for Gln over a 30-fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed r ole of RNA as a cofactor in optimizing amino acid activation suggests that the tRNA(Gln)-GlnRS complex may be partly analogous to ribonucleo protein enzymes where protein-RNA interactions facilitate catalysis.