Multisite-specific archaeosine tRNA-guanine transglycosylase (ArcTGT) from Thermoplasma acidophilum,a thermo-acidophilic archaeon

Archaeosine (G⁺), which is found only at position 15 in many archaeal tRNA, is formed by two steps, the replacement of the guanine base with preQ₀ by archaeosine tRNA-guanine transglycosylase (ArcTGT) and the subsequent modification of preQ₀ to G⁺ by archaeosine synthase. However, tRNA^Leu from Thermoplasma acidophilum, a thermo-acidophilic archaeon, exceptionally has two G⁺13 and G⁺15 modifications. In this study, we focused on the biosynthesis mechanism of G⁺13 and G⁺15 modifications in this tRNA^Leu. Purified ArcTGT from Pyrococcus horikoshii, for which the tRNA recognition mechanism and structure were previously characterized, exchanged only the G15 base in a tRNA^Leu transcript with ¹⁴C-guanine. In contrast, T. acidophilum cell extract exchanged both G13 and G15 bases. Because T. acidophilum ArcTGT could not be expressed as a soluble protein in Escherichia coli, we employed an expression system using another thermophilic archaeon, Thermococcus kodakarensis. The arcTGT gene in T. kodakarensis was disrupted, complemented with the T. acidophilum arcTGT gene, and tRNA^Leu variants were expressed. Mass spectrometry analysis of purified tRNA^Leu variants revealed the modifications of G⁺13 and G⁺15 in the wild-type tRNA^Leu. Thus, T. acidophilum ArcTGT has a multisite specificity and is responsible for the formation of both G⁺13 and G⁺15 modifications.