Figure 4.

In vitro guanine exchanging activities of purified ArcTGT and S-30 fraction from T. acidophilum. (A) Cloverleaf structure of tRNA^Leu_UAG from T. acidophilum. Two G⁺ (G⁺13 and G⁺15) modifications are present in this tRNA. Abbreviations of other modified nucleosides are as follows: 4-thiouridine, s⁴U; N², N²-dimethylguanosine, m²₂G; 5-carbamoylmethyuridine, ncm⁵U; N¹-methylguanosine, m¹G; 7-methylguanosine, m⁷G; pseudouridine, Ψ; 2′-O-methylcytidine, Cm; N¹-methyladenosine, m¹A. (B) Possibility of structural change in the D-arm of tRNA^Leu. (C) The cloverleaf structures of wild-type and mutant tRNA^Leu transcripts. The G13 and/or G15 in the wild-type tRNA^Leu were substituted by A in mutant tRNA^Leu transcripts. The nucleosides at positions 13 and 15 are highlighted in black. (D) In vitro guanine exchanging activity of purified ArcTGT and T. acidophilum S-30 fraction. To verify whether the purified ArcTGTs and S-30 fraction from T. acidophilum exchange the guanine base at position 13, ArcTGTs from P. horikoshii (left) and T. kodakarensis (middle) were purified and the S-30 fraction was prepared from T. acidophilum cells (right). The proteins were analyzed by SDS-PAGE. The gels were stained with Coomassie Brilliant Blue. ¹⁴C-guanine exchanging activities were tested using these proteins and tRNA^Leu transcripts. After the reaction, tRNA transcripts were extracted with phenol-chloroform, recovered by ethanol precipitation and separated by 10% PAGE (7 M urea). The RNAs were visualized by staining with methylene blue. Autoradiograms of the gels were acquired. (E) Time-course experiments of ¹⁴C-guanine exchanging activity in T. acidophilum S-30 fraction were performed. The samples were taken at 0, 5, 10 and 20 min-periods and loaded onto 10% polyacrylamide gels, which contained 7 M urea. The RNAs were visualized by methylene blue staining and autoradiograms of the gels were acquired.