Analysis of 3′-amino-tailed tRNA by aminoacylation and alkali hydrolysis. (A) Levels of stably charged Pro-tRNAPro after alkali hydrolysis in 100 mM glycine, pH 9.0, for 3.5 h at 37°C. In each protocol, the 3′-amino-tailed Ec tRNAPro was charged with 3H-Pro by the cognate ProRS, followed by alkali hydrolysis, ethanol precipitation, and measured for counts by a scintillation counter. The amount of label was measured before (−) and after (+) alkali hydrolysis and shown as the percentage of input tRNAPro. For proline aminoacylation, charging by ProRS was usually in the range of 8%–9% as shown in the –pH 9.0 column. (B) The level of alkali-resistant Pro-tRNAPro was determined from A by measuring the ratio of post-alkali counts (+pH 9.0) over pre-alkali counts (−pH 9.0). These data show that protocol 2 produces the highest level of 3′-amino-tailing. Errors are standard deviation and n = 3.
