FIGURE 6.

Test of the putative Nudix hydrolase HP0507 for RNA pyrophosphohydrolase activity. In vitro transcribed A8XL RNA radiolabeled at the 5′-terminal γ-phosphate and internally labeled with fluorescein (see Fig. 3) was treated with purified HpRppH (2 nm final concentration), catalytically inactive HpRppH-E57Q (2 nm), or HP0507 (2 or 20 nm), and reaction samples quenched at time intervals were subjected to gel electrophoresis. Hydrolytic release of the 5′-terminal radiolabel was detected by autoradiography (P-32), and the integrity of the remainder of the RNA molecule was monitored by fluorescence (Fluor).