tRNA recognition of tRNA-guanine transglycosylase from a hyperthermophilicarchaeon, Pyrococcus horikoshii

In the biosynthesis of archaeosine, archaeal tRNA-guanine 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 TG T from a hyperthermophilic archaeon, Pyrococcus horikoshii. Mutational stud ies using variant tRNA(Val) transcripts revealed that both guanine and its location (position 15) were strictly recognized by TGT without any other se quence-specific requirements. It appeared that neither 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 jun ction between the two domains served as a good substrate, suggesting that m ismatched nucleotides in the helix provide the specific information that al lows TGT to recognize the guanine in the D loop. Our findings indicate that the tRNA recognition requirements of P. horikoshii TGT are sufficiently li mited and specific to allow the enzyme to recognize efficiently any tRNA sp ecies whose structure is not fully stabilized in an extremely high temperat ure environment.