Molecular Mechanism of tRNA Recognition by Aminoacyl-tRNA Synthetase from Hyperthermophilic Achaeon

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To investigate the molecular recognition and discrimination mechanism of proline, tyrosine, histidine and tryptophan tRNA by each aminoacyl-tRNA synthetase (ARS) from hyperthermophilic and aerobic crenarchaeon, Aeropyrum pernix K1, each synthetase (ProRSAP, TyrRSAP, HisRSAP and TrpRSAP, respectively) gene was cloned, overexpressed and purified by using several purification procedures including affinity chromatography. Each amino acid specific wild type and mutant tRNA variants were prepared by in vitro transcription system with T7 RNA polymerase. These transcripts were aminoacylated with each amino acid by the recombinant ARS. ProRSAP recognized G35 and G36 of anticodon, discriminator base A73 and G1-C72 base pair at acceptor end. This G1-C72 specific recognition of acceptor end is different from Escherichia coli recognition site G72. This results suggest that the difference of recognition mechanism within A. pernix and E. coli ProRS may caused to change of nucleotides at position 1 an … More d 72 of proline tRNA during evolution. With respect to the long variable arm in E. coli tyrosine tRNA, some base pairs in length was required for tyrosylation. None of the recognition sites were found in the acceptor stem, except the discriminator base A73 in E. coli system. Archaeal and eukaryotic tyrosine tRNA have a unique C-G72 base pair. TyrRSAP did recognize this first C1-G72 pair in addition to the discriminator base A73 in the acceptor region and anticodon nucleotides. All known histidine tRNAs have an additional nucleotide at the 5' end compared with the other tRNA species. A. pernix histidine tRNA based on the information from database shows that A. pernix histidine tRNA does not have minus 1G residue. This additional minus 1G was strongly recognized by the HisRSAP. Surprisingly, minus IA and minus 1C histidine tRNA mutants also showed high histidylation activity. Discriminator base C73 was weekly recognized. HisRSAP does not recognize the anticodon similar to that of E. coli recognition system. HisRSAP can also recognize nonsense histidine tRNA. E. coli tryptophan tRNA is base-specifically recognized acceptor stem Al-U72, G2-C71 and G3-C70 base pairs, except anticodon nucleotides and discriminator base G73. TrpRSAP recognized all anticodon nucleotides C34, C35 and A36, discriminator base A73, G1-C72 and G2-C71 base pairs of acceptor stem. These results indicate that the recognition sites in the acceptor stem including discriminator base are so different between A. pernix and E. coli system. Less