Wild-type TbADAT2/3 specifically binds tRNA in vitro. (A) Representative electrophoretic mobility shift assay (EMSA) to determine the extent of tRNA binding in the presence of either nonradioactive tRNA or a nontRNA (spliced leader RNA) substrate used as specific and nonspecific competitors, respectively. The left panel shows TbADAT2/3 binding to radioactive tRNAVal in the presence of the same nonlabeled tRNA. The right panel shows TbADAT2/3 binding to the same radioactively labeled tRNAVal but in the presence of splice leader RNA. Lane 1 shows a mock reaction in which the tRNA probe was incubated in binding buffer in the absence of enzyme and competitor. Lane 2 shows the tRNA probe incubated with wild-type enzyme in the absence of any competitors. Lanes 3–7 and 8–12 show tRNA incubated with an increasing excess of cold competitor (2-, 4-, 8-, 16-, and 64-fold). “Free probe” denotes the migration of the unbound tRNA, and “complex” denotes the migration of the TbADAT2/3 bound tRNA. (B) The reaction products from A were used to calculate the fraction of tRNA bound by calculating the percent of the RNA shifted divided by the total (bound and unbound) probe in each reaction. These values were plotted against competitor fold-excess and fitted to a single exponential decay curve using SigmaPlot.
