Aminoacylation Assay for Chiral Amino Acid Systems

In vitro aminoacylation assay was conducted according to the previous papers^{67 (link),68 (link)}. For chimeric Phe and Ala system, 100 nM synthetases were incubated with 0, 30 μg of extracted tRNAs in reaction buffer (50 mM Tris–HCl, 10 mM MgCl₂, 20 mM KCl, 2 mM DTT, pH 7.4) supplemented with 4 mM ATP, 0.1 mg/ml BSA and 80 μM L-phenylalanine, 17.8 μM L-[¹⁴C]-phenylalanine for chimeric Phe system or 50 μM L-alanine, 33.3 μM L-[¹⁴C]-alanine for chimeric Ala system at 37 °C for 40 min. After incubation, the aliquots were spotted onto Whatman GF/C glass microfiber filter pre-equilibrated with 10% cold TCA. The paper filters were washed with cold 10% TCA four times and then 95% ethanol four times. After washing, the filter papers were visualised by the liquid scintillation counter to quantify the radioactivity. For chimeric His system, 25 nM synthetase was incubated with 0, 10 and 30 μg of extracted tRNAs in reaction buffer (50 mM Tris–HCl, 10 mM MgCl₂, 20 mM KCl, 2 mM DTT, pH 7.4) supplemented with 0.1 mM ATP, 1 mM L-histidine and 0.1 mg/ml BSA at 37 °C for 2 h. The aminoacylation was measured through the production of AMP with AMP-Glo assay (Promega) according to the manufacturer’s instructions.