Effects of arginine mutations on anticodon nuclease activity in vitro. (A) Aliquots (1 μg) of the recombinant wild-type (WT) and Arg-to-Ala mutant γ-toxin polypeptides as specified were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (in kDa) of marker polypeptides are indicated on the left. Anticodon nuclease reaction mixtures contained 200 fmol 5′ 32P-labeled 17-mer stem–loop RNA and 200 fmol of the indicated γ-toxin preparations. The extents of RNA cleavage are plotted. (B) Conservative mutations of Arg84 and Arg151. Aliquots (1 μg) of the WT and mutant γ-toxin polypeptides as specified were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown in the left panel. Anticodon nuclease reaction mixtures contained 200 fmol 5′ 32P-labeled 17-mer stem–loop RNA and 500 fmol of the indicated γ-toxin preparations. The extents of RNA cleavage are shown.
