ISSN:
1617-4623
Keywords:
Key words Glutaminyl-tRNA synthetase
;
Temperature sensitivity
;
Substrate specificity
;
Aminoacylation
;
Suppressor tRNA
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract It has previously been shown that the single mutation E222K in glutaminyl-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 investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNAGln : 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 tRNAGln. The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischarge supF tRNATyr 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 distinguish supE tRNAGln CUA from tRNAGln 2 considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher Km 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF02173978
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