The UPF1-NKR mutant protein fails to bind RNA and hydrolyze ATP in vitro. (A) Coomassie-stained gel showing the purified UPF1-3xFLAG WT, DE, and NKR mutants and MBP-3xFLAG purified as a control. A BSA standard curve loaded on the same gel was used to estimate the concentration of the purified proteins. (M) Molecular weight marker. (B) In vitro RNA binding was analyzed by MicroScale Thermophoresis (MST). Binding reactions were incubated for 1 h at 25°C in the absence or presence of ATP, followed by MST. The fraction of bound RNA is plotted against the final concentration of the unlabeled titrated proteins, and the averages and standard deviations from three independent dilution series of UPF1 are shown. Estimated dissociation constants (Kd) are shown for UPF1-WT and UPF1-DE proteins; (n/a) not available. (C) NADH-coupled ATPase assays were performed for 2 h at 37°C, and the oxidation of NADH was followed by measuring absorbance at 340 nm. Shown are the averages and standard deviations of four to six independent measurements.
