FIGURE 5.
Time and nATP dependence of tRNA 3′-amino-tailing in protocol 2. (A) Denaturing 12% PAGE/7 M urea gel analysis of 3′-amino-tailing of Ec tRNAPro over time in protocol 2. After biotin-streptavidin conjugation, the tRNA (70 ng) was analyzed by denaturing PAGE. (B) The level of 3′-amino-tailed tRNAPro analyzed from A is plotted over time, showing a plateau of tailing at 20–30 min. (C) Extent of 3′-amino-tailing of Ec tRNAPro as a function of nATP concentration in the presence or absence of 1 mM PPi. Reactions were incubated 60 min at 37°C using equimolar concentrations of tRNA and the CCA enzyme and analyzed as described above. Errors are standard deviation and n = 3.
