The RNA-binding PUA Domain of Archaeal tRNA-Guanine Transglycosylase Is Not Required for Archaeosine Formation
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA. Journal of Biological Chemistry
(Impact Factor: 4.57).
04/2006; 281(11):6993-7001. DOI: 10.1074/jbc.M512841200
Bacterial tRNA-guanine transglycosylase (TGT) replaces the G in position 34 of tRNA with preQ1, the precursor to the modified nucleoside queuosine. Archaeal TGT, in contrast, substitutes preQ0 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 kcat (2.8 × 10–3 s–1) and results in a 75-fold increased Km for tRNAAsp(1.2 × 10–5 m) compared with full-length TGT. Mutations in tRNAAsp altering or abolishing interactions with the PUA domain can compete with wild-type tRNAAsp 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.
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