Mn2+ stimulates recombinant yeast decapping activity. (A) Decapping reactions were carried out by incubating 300 ng of the indicated histidine-tagged Dcp1p, Dcp2ΔCp, or Dcp2ΔCpQ153E proteins with cap-labeled pcP-G16RNA for 30 min at 37°C in our standard reaction buffer (Mg2+ only) or buffer supplemented with 2 mM MnCl2(Mg2+ and Mn2+). Standards were simultaneously developed and their positions are denoted on the right of the panel. (B) Gel-isolated Dcp2p is enzymatically active. Three micrograms of His-Dcp2 ΔC and Dcp2ΔC Q153E3 were resolved by a standard SDS/PAGE and visualized by Coomassie blue staining (left panel) or resolved in an identical gel containing cap-labeled pcP-G16 RNA and renatured and assayed for decapping activity (right panel) (see Materials and Methods). Protein size markers are indicated between the gels.
