Figure 8

RNase activity assays with purified VapD, Cat, and VapX. Ribonuclease activity over time of the protein elution buffer control (blue), 0.2 μg (red), 0.4 μg (green), and 0.6 μg (purple) of purified VapD, 0.6 μg of chloramphenicol acetyltransferase (Cat, turquoise), or 0.6 μg of VapX (orange) assayed with the RNaseAlert test kit (IDT, Coralville, IA). Indicated amounts of proteins were added to 25 pmol fluorophore-conjugated RNaseAlert substrate. The substrate has a quencher on one end and a fluorophore (FAM) on the other. Cleavage of the single-stranded RNA removed the quencher and the resulting fluorescence was read on a MiniOpticon real-time detection system. The Cat protein and the VapX antitoxin were overexpressed and purified in an identical fashion to VapD and serve as negative protein controls.