Bacterial tRNA-guanine transglycosylase (TGT) replaces the G in position 34 of tRNA with preQ(1), the precursor to the modified nucleoside queuosine. Archaeal TGT, in contrast, substitutes preQ(0) for the G in position 15 of tRNA as the first step in archaeosine formation. The archaeal enzyme is about 60% larger than the bacterial protein; a carboxyl-terminal extension of 230 amino acids contains the PUA domain known to contact the four 3'-terminal nucleotides of tRNA. Here we show that the C-terminal extension of the enzyme is not required for the selection of G15 as the site of base exchange; truncated forms of Pyrococcus furiosus TGT retain their specificity for guanine exchange at position 15. Deletion of the PUA domain causes a 4-fold drop in the observed k(cat) (2.8 x 10(-3) s(-1)) and results in a 75-fold increased K(m) for tRNA(Asp)(1.2 x 10(-5) m) compared with full-length TGT. Mutations in tRNA(Asp) altering or abolishing interactions with the PUA domain can compete with wild-type tRNA(Asp) for binding to full-length and truncated TGT enzymes. Whereas the C-terminal domains do not appear to play a role in selection of the modification site, their relevance for enzyme function and their role in vivo remains to be discovered.