Purification and Enzymatic Digestion of tRNA

Transfer RNA was purified from K12 strain of E. coli using Tri-reagent by differential precipitation with sodium salts as described before.^{63 (link)} Isolated tRNA was reprecipitated with 3M ammonium acetate to replace the sodium ions at the final purification step before dissolving it in water. RNase T1 and bacterial alkaline phosphatase were procured from Worthington Biochemical Corporation. The RNases MC1^{62 (link)} and cusativin^{61 (link)} were overexpressed in BL21 and Rosetta™ (DE3) (Novagen) cells, respectively, and purified on a nickel column using His-tag protein purification kit (EMD Millipore) as per the manufacturer’s guidelines. Each batch of ribonuclease protein preparation was evaluated for optimal RNA:protein ratio to ensure appropriate digestion of substrate RNA. In general, optimal digestion of the tRNA mixture or rRNA was carried out by mixing 0.5–1.0 μg of ribonuclease protein for each μg of RNA in the presence of 120 mM ammonium acetate (pH not adjusted) for 90 min at 37 °C for MC1 and 62 °C for cusativin.