Identity elements ofThermus thermophilustRNAThr

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Abstract
In this study, we identified nucleotides that specify aminoacylation of tRNAThr by Thermus thermophilus threonyl-tRNA synthetase (ThrRS) using in vitro transcripts. Mutation studies showed that the first base pair in the acceptor stem as well as the second and third positions of the anticodon are major identity elements of T. thermophilus tRNAThr, which are essentially the same as those of Escherichia coli tRNAThr. The discriminator base, U73, also contributed to the specific aminoacylation, but not the second base pair in the acceptor stem. These findings are in contrast to E. coli tRNAThr, where the second base pair is required for threonylation, with the discriminator base, A73, playing no roles. In addition, among several mutations at the third base pair in the acceptor stem, only the G3-U70 mutant was a poor substrate for ThrRS, suggesting that the G3-U70 wobble pair, which is the identity determinant of tRNAAla, acts as a negative element for ThrRS. Similar results were obtained in E. coli and yeast. Thus, this manner of rejection of tRNAAla is also likely to have been retained in the threonine system throughout evolution.