FIGURE 5.
The C terminus of TbADAT2 is essential for tRNA binding. Two mutants were generated bearing either a deletion of the last 10 amino acids of TbADAT2 (ADAT2 C-terΔ10) or a replacement of the five positively charged amino acids of the KR-domain by alanines (ADAT2 C-ter5A). These TbADAT2 mutants were co-expressed with wild-type TbADAT3 in E. coli, and the resulting recombinant proteins (heterodimers) were purified by Ni2+-chelate chromatography. These mutants were used in EMSA assays. (A) Radioactive G34-containing tRNAVal was incubated with increasing concentrations of recombinant ADAT2 C-terΔ10/TbADAT3 heterodimer (Materials and Methods). (B) A similar experiment as in A but with ADAT2 C-ter5A. “Free probe” denotes the migration of the unbound tRNA, and “complex” denotes the migration of the protein-bound tRNA, also highlighted by arrows.