Study on tRNA recognition mechanism by aminoacyl-tRNA synthetase from archaea

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Although the tRNA identity elements have been widely investigated in eubacterial system including Escherichia coli, little is known about tRNA recognition sites for aminoacyl-tRNA synthetase (ARS) from archaea, which is classified as a third kingdom. To study the molecular mechanism of recognition of threonine, proline and tryptophan tRNA for each ARS from extreme thermophilic archaeon, Aeropyrum pernix K1, each ARS gene was cloned and overexpressed. Aminoacylation experiments using in vitro wild and mutant tRNA transcripts and overexpressed ARSs were examined. In the threonine system, the results support our previous finding that A.pernix ThrRs does not recognize the discriminator base of threonine tRNA. This result indicated the diversity of importance of the discriminator base among archaea because the discriminator base of threonine tRNA from halophilic archaeon, Haloferax volcanii, is a strong determinant by cognate ThrRS. It was also found that A.pernix ThrRs did recognize the ac … More ceptor stem region of threonine tRNA as well as other species ThrRS, in spite of N-terminal truncated unique structure. Furthermore, it was shown that in vitro transcript of proline tRNA whose second and third pairs of acceptor stem was mutated to C-G in addition to transplantation of G36U was threonylated by A.pernix ThrRS. This result of switching from proline tRNA to threonine tRNA by transplanting only five bases indicates that the major identity elements of A.pernix threonine tRNA are acceptor stem region and anticodon. In the proline system, it was found that the discriminator base A73, G1-C72 base pair, and anticodon G35 and G36 were strong determinants for A.pernix ProRs. Recognition of G1-C72 base pair, and anticodon G35 and G36 were strong determinants for A.pernix ProRS. Recognition of G1-C72 base pair was quite different from E.coli ProRS system. In the tryptophan system, results showed that anticodon C34, C35 and A36, discriminator base G73, G1-C62 and G2-C71base pairs of acceptor stem end were base specifically recognized by A.pernix TrpRS. These results indicate that the tRNA recognition mechanisms by ARS is different among three kingdomes. Less