Unique recognition style of tRNA(Leu) by Haloferax volcanii leucyl-tRNA synthetase

The recognition manner of tRNA(Leu), a class II tRNA characterized by a long variable arm, by leucyl-tRNA synthetase from an extreme halophilic archaea, Haloferax volcanii, was studied using the in vitro transcription system. It was found that the discriminator base (A73) and the long variable arm, especially the specific loop sequence A47CG47D and U47H at the base of this helix, are significant for recognition by LeuRS. An appropriate stem length of the variable arm was also required. Base substitutions in the anticodon arm did not affect the leucylation activity. Transplantation of both the discriminator base and the variable arm of tRNA(Leu) was not sufficient to introduce leucylation activity to tRNA(Ser). Insertion of an additional nucleotide into the D-loop, which is not involved in the direct interaction with LeuRS, converted tRNA(Ser) to an efficient leucine acceptor. This suggests that differences in the tertiary structure play a key role in eliminating tRNA(Ser). The sequence-specific recognition of the long variable arm of tRNA(Leu) has not been observed in any of other organisms reported, such as Escherichia coli, yeast or human. On the other hand, the mode of discrimination from non-cognate tRNAs is similar to that in E. coli in that differences in the tertiary structure play a key role. Similarity extends to the substrate stringency, exemplified by a cross-species aminoacylation study showing that no class II tRNAs from E. coli or yeast can be leucylated by H. volcanii LeuRS. Our results have implications for the understanding of the evolution of the recognition system of class II tRNA.