The procedures for cloning, expression and purification of proteins and protein complexes used for in vitro studies (enzymology, SEC-MALLS and X-ray crystallography) are described in supplementary materials . The tRNAs from S. cerevisiae or H. volcanii strains were purified as previously described (47 (link)).
The MTase assays were performed in a total volume of 10 μl (for HvoMtq2-Trm112) or 20 μl (for HvoTrm9–Trm112) containing 400 mM potassium phosphate buffer pH 7.5, 3 M KCl, 2.5 mM EDTA, 5 mM MgCl2, 5 mM NH4Cl, 0.25 mg/ml Bovine Serum Albumin, 50 μM S-adenosyl-l -methionine (SAM; containing 0.87 Ci/mmol of [3H]-SAM; Perkin Elmer) and 5 pmol of HvoMtq2–Trm112 complex or 2 pmol of HvoTrm9–Trm112 complex. The reaction was initiated by adding 100 pmol of substrate (HvoaRF1, HvoaRF3 or total tRNAs) to the mixture. After incubation for 2 h at 45°C, the reaction was stopped by precipitation with cold trichloroacetic acid (5%), followed by filtration on Whatman GF/C filters. The [3H] incorporation was measured using a Beckman Coulter LS6500 scintillation counter.
The MTase assays were performed in a total volume of 10 μl (for HvoMtq2-Trm112) or 20 μl (for HvoTrm9–Trm112) containing 400 mM potassium phosphate buffer pH 7.5, 3 M KCl, 2.5 mM EDTA, 5 mM MgCl2, 5 mM NH4Cl, 0.25 mg/ml Bovine Serum Albumin, 50 μM S-adenosyl-
