Discrimination of tRNALeu Isoacceptors by the Mutants of Escherichia coli Leucyl-tRNA Synthetase in Editing

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Abstract
Leucyl-tRNA synthetase (LeuRS), one of the class Ia aminoacyl-tRNA synthetases, joins Leu to tRNALeu and excludes noncognate amino acids in protein synthesis. In this study, Escherichia coli LeuRS mutants at amino acid E292, which was located in the connective polypeptide 1 insertion region, were synthesized. Although mutated LeuRS showed little change in structure compared with wild-type LeuRS, the mutants were impaired in activity to varying extents. It was also showed that mutations did not affect the adenylation reaction. However, mutated LeuRS can mischarge tRNALeu isoacceptors tRN or tRN with isoleucine to different extents. Isoleucylation of tRN was more than that of tRN . The mutant LeuRS-E292S, which was picked out as an example for the investigation of the relationship between tRNALeu isoacceptors and editing function, can discriminate the Watson−Crick base pair of the first base pair of tRNALeu from the wobble base pair. The tRNALeu with the Watson−Crick base pair may result in more isoleucylated product than that with the wobble base pair. The same phenomenon happened to another mutant, LeuRS-A293D. It seems that the flexibility of the first base pair affects the editing reaction of LeuRS. The results indicate that the flexibility of the first base pair of tRNALeu may probably affect the mischarged 3‘-end of tRNALeu shuttling from synthetic site to editing site and that the transferred acceptor arm of tRNALeu may interact with LeuRS in the region around E292.

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