GENETIC-ANALYSIS OF FUNCTIONAL CONNECTIVITY BETWEEN SUBSTRATE RECOGNITION DOMAINS OF ESCHERICHIA-COLI GLUTAMINYL-TRANSFER-RNA SYNTHETASE

It has previously been shown that the single mutation E222K in glutami nyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype on Escherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to inv estigate the role of these residues in substrate discrimination. The t hree-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 intera ctions was confirmed by the observation that GlnRS-E222K was able to m ischarge supF tRNA(Tyr) considerably more efficiently than the wild-ty pe enzyme, whereas GlnRS-E222K/C171G could not. These differences in s ubstrate specificity also extended to anticodon recognition, with the double mutant able to distinguish supE tRNA(CUA)(Gln) from tRNA(2)(Gln ) considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E 222K was found to have a 15-fold higher K-m for glutamine than the wil d-type enzyme, whereas the double mutant only showed a 7-fold increase . These results indicate that the C171G mutation improves both substra te discrimination and recognition at three domains in GlnRS-E222K, con firming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.