UV melting experiments

Pre-tRNAs were folded as described above before each experiment using a “UV melting buffer” of the final concentration of 10 mM PIPES pH = 7, 150 mM NaCl, 1 mM MgCl₂, and 1 µM pre-tRNA. Thermodynamic curves were obtained by heating up the samples 1°C/min from 15°C to 95°C while absorbance at 260 nm was monitored at every 0.5°C or 1°C using a Cary 300 spectrophotometer. Data were corrected with data from blank measurements and analyzed with fitUVData.py and Global Melt Fit (Draper et al. 2000; Mustoe et al. 2015). Most pre-tRNA showed three or four transitions in agreement with previous findings (Mustoe et al. 2015). Average T_ms and standard errors were calculated from at least three independent experiments, and bootstrapping analysis was performed to ensure the robustness of the data where it was applicable.