FIGURE 3.

Mutations of highly conserved residues in the HEPN domain affect RNase activity. (A) Radiolabeled ssRNA (37mer A) was incubated with no protein for 30 min (−), with wild-type (wt) or mutant Csx1 for 1 min (1) or for 30 min (30), followed by separation by denaturing gel electrophoresis. The arrow indicates the full-length RNA, while the bracket indicates Csx1 cleavage products. (B) Purified wt and mutant Csx1 proteins were analyzed by SDS-PAGE and Coomassie blue staining. Molecular weight marker is indicated in kilodaltons. (C) Csx1 cleavage activity occurs in the absence of added metal ions (−EDTA) and in the presence of a wide range (0.5, 1, 200, 500, 1000 µM) of EDTA. The dotted line separates data that was subject to longer exposure times to visualize molecular weight markers.