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J. Biol. Chem., Vol. 281, Issue 11, 6993-7001, March 17, 2006

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The RNA-binding PUA Domain of Archaeal tRNA-Guanine Transglycosylase Is Not Required for Archaeosine Formation^*

From the Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114

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 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.

Received for publication, December 1, 2005 , and in revised form, January 10, 2006.

^* This work was supported by a grant GM022854 from NIGMS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 203-432-6200; Fax: 203-432-6202; E-mail: soll{at}trna.chem.yale.edu.

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