Effects of glutamate mutations on anticodon nuclease activity in vitro. (A) Aliquots (2 μg) of the recombinant wild-type (WT) and mutant γ-toxin polypeptides as specified were analyzed by SDS-PAGE (15% acrylamide). The Coomassie blue-stained gel is shown. The positions and sizes (in kDa) of marker polypeptides are indicated on the left. Below the figure is a synopsis of the toxicity (+) or lack thereof (−) of the WT and mutant proteins when expressed in vivo in yeast (Keppetipola et al. 2009). (B) Anticodon nuclease reaction mixtures contained 200 fmol 5′ 32P-labeled R17 stem–loop and 100 fmol of the indicated γ-toxin preparations. The products were analyzed by denaturing PAGE, and the extents of RNA cleavage were quantified.
