AMINOACYL-TRANSFER-RNA SYNTHETASES OPTIMIZE BOTH COGNATE TRANSFER-RNARECOGNITION AND DISCRIMINATION AGAINST NONCOGNATE TRANSFER-RNAS

Specific protein-nucleic acid interactions are usually the product of sequence-dependent hydrogen bonding. However, in the crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex wit h tRNA(Gln), leucine 136 (Leu136) stabilizes the disruption of the wea k first (U1-A72) base pair in tRNA(Gln) by stacking between A72 and G2 . We have demonstrated, by a combined in vivo and in vitro mutational analysis, that Leu136 is important for tRNA specificity despite making no hydrogen bonds with tRNA(Gln). Both more (L136F) and less (L136V, L136M, L136A, and L136T) mischarging mutants of GlnRS have been identi fied. GlnRS(L136F) is more mischarging and less specific than wild-typ e GlnRS in vivo, due not to an increased affinity for the noncognate t RNAs but to a decreased affinity for tRNA(Gln). Also, unlike other mis charging mutants of GlnRS that have been characterized, it does not ex hibit generally relaxed tRNA specificity in vivo and mischarges only a subset of the tRNAs tested. A possible sequence preference for a Py1- Pu72/Pu2-Py71 combination is suggested. The L136A/M/T/V mutants are th e first GlnRS variants, including wild-type, expressed on pBR322 which no longer mischarge tyrT(UAG) in vivo. We have shown that, while the L136A mutant is less mischarging than wild-type both in vivo and in vi tro, it is not more specific as it also exhibits reduced affinity for its cognate glutamine tRNA. On the basis of these results, we suggest that the aminoacyl-tRNA synthetases have evolved to balance cognate tR NA recognition and discrimination against noncognate tRNAs.