Figure 5.

Acid gel analysis of tRNA^leu-ΔACVL1 mutant. A73C depicts the discriminator base change from A73 to C73. G18A:U55C, G19A:G56U and G18A:U55C/G19A:G56U are notations for the mutational changes G18:U55 to A18:C55, G19:C56 to A19:U56 and a double mutant with both G18:U55 and G19:C56 changed to A18:C55 and A19:U56, respectively. Each aminoacylation reaction was performed with 60 µM RNA, 20.5 µM ¹⁴C-leucine (50 µCi/ml) and 100 nM LeuRS under the conditions described in Materials and Methods. (A) RNA products were detected by ethidium bromide staining of a native acidic gel. An extra lane without loaded RNA (lane 2) separates assays for tRNA^leu-ΔACVL1 from each of its mutant variants. (B) RNA products aminoacylated with ¹⁴C-leucine were visualized by phosphoroimaging the dried acidic gel for two weeks. (C) Denatured RNA products were detected by ethidium bromide staining of a denaturing acidic polyacrylamide gel. (D) Denatured ¹⁴C-leucine aminoacylated products were visualized by phosphoroimaging the dried acidic gel for two weeks.