The RNA-binding PUA domain of archaeal tRNA-guanine transglycosylase is not required for archaeosine formation.
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.
Sabina
Jeffrey
J
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Söll
Dieter
D
D001105:Archaea / Q000201:enzymology*; D020134:Catalytic Domain; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D017353:Gene Deletion; D006147:Guanine / Q000737:chemistry; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010430:Pentosyltransferases / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000378:metabolism; D012313:RNA / Q000737:chemistry*; D012343:RNA, Transfer / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014158:Transcription, Genetic
D017931:DNA Primers; D009841:Oligonucleotides; D006151:Guanosine; C081066:archaeosine; D006147:Guanine; D012313:RNA; D012343:RNA, Transfer; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase