Genetic analysis of functional connectivity between substrate recognition domains ofEscherichia coli glutaminyl-tRNA synthetase

It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype onEscherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNA^Gln: GlnRS:ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3′ end of tRNA^Gln. The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischargesupF tRNA^Tyr considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not. These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguishsupE tRNA _CUA ^Gln from tRNA ₂ ^Gln considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher K_m for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase. These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.