In the biosynthesis of archaeosine, archaeal tRNAguanine transglycosylase (TGT) catalyzes the replacement of guanine at position 15 in the D loop of most tRNAs by a free precursor base. We examined the tRNA recognition of TGT from a hyperthermophilic archaeon, Pyrococcus horikoshii. Mutational studies using variant tRNA Val transcripts revealed that both guanine and its location (position 15) were strictly recognized by TGT without any other sequence-specific requirements. It appeared that

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... er the global L-shaped structure of a tRNA nor the local conformation of the D loop contributed to recognition by TGT. A minihelix composed of the acceptor stem and D arm of tRNA Val , designed as a potential minimal substrate, failed to serve as a substrate for TGT. Only a minihelix with mismatched nucleotides at the junction between the two domains served as a good substrate, suggesting that mismatched nucleotides in the helix provide the specific information that allows TGT to recognize the guanine in the D loop. Our findings indicate that the tRNA recognition requirements of P. horikoshii TGT are sufficiently limited and specific to allow the enzyme to recognize efficiently any tRNA species whose structure is not fully stabilized in an extremely high temperature environment.