EV71 2C^ATPase possesses both ATP-dependent and ATP-independent helix unwinding activities.

<p><b>(A)</b> Schematic illustration of the standard RNA helix substrate that contains both 3′- and 5′-protrusions. <b>(B)</b> and <b>(C)</b> The standard RNA helix (0.1 pmol) was reacted with MBP-2C^ATPase in the absence or presence of the indicated NTPs (5 mM) (B) or in the presence of increasing concentrations (0–6 mM) of ATP as indicated (C). <b>(D)</b> The unwinding activities under different ATP concentrations were plotted as the percentage of the released RNA from the total RNA helix substrate (Y-axis) at each ATP concentration (X-axis). Error bars represent standard deviation (SD) values from three separate experiments. <b>(E)</b> Unwinding assays of 2C^ATPase using the standard helix substrate were performed in the absence or presence of ATP or ATP analog (AMP-PNP) as indicated. <b>(F)</b> The ATPase activity of MBP-2C^ATPase was measured as nanomoles of released inorganic phosphate at the indicated Mg²⁺ concentrations. Error bars represent SD values from three separate experiments. <b>(G)</b> and <b>(H)</b> Unwinding assays of 2C^ATPase were performed in the presence of 0–2.5 mM (G) or 3–20 mM (H) Mg²⁺ as indicated. <b>(I)</b> The ATPase activity of MBP-2C^ATPase was measured as nanomoles of released inorganic phosphate at the indicated GnHCl concentrations. Error bars represent SD values from three separate experiments. <b>(J)</b> Unwinding assays of 2C^ATPase were performed in the presence of 0–50 mM GnHCl as indicated.