Figure 2.

CCA-adding enzyme-catalyzed CCA incorporation on tRNA^His lacking G-1 (tRNA^HisΔG-1; A) and tRNA^His+G-1 (B) from S. cerevisiae. tRNA variants were incubated with increasing amounts of CCA-adding enzyme (0.5, 1.5, 3.0, 4.5, 6.0, 60 und 300 ng). The reaction products were separated on a denaturing polyacrylamide gel and visualized by ethidium bromide staining. Both tRNA variants were processed at comparable efficiencies, resulting in a complete substrate turnover. NC, negative control without enzyme. (C) Kinetic analysis of CCA-addition for both tRNA variants. Increasing amounts of tRNA^HisΔG-1 (black curve) and tRNA^His+G-1 (green curve) were incubated with CCA-adding enzyme, NTPs and α-³²P-ATP under steady-state conditions. Michaelis–Menten data were calculated from triplicates using GraphPadPrism software (Table 1).