The length of the ssRNA 5′ overhang influences helicase and ATPase activity of DbpA. (A) RNA constructs used for activity assays. The substrate duplex (orange) is located 5′ to HP92 (red). The varying length of the 5′ overhangs is indicated. For helicase assays, the 9mer RNA contained a fluorescein label at the 5′ end (indicated by a green star). (B) Unwinding rates observed in single-turnover experiments are plotted versus the 5′ overhang length. Results from three measurements are shown. (C) ATPase turnover rates are plotted versus the 5′ overhang length. Rates were determined in the absence (gray) and presence of the 9mer RNA (red). Results from three measurements are shown. (D) The number of hydrolyzed ATP molecules for each unwinding event are plotted versus the 5′ overhang length. The values represent mean and standard deviation calculated from the experiments shown in panels B and C. (E) Single-turnover unwinding of RNA constructs with a 5′ overhang of 2 nt (red) or 8 nt (blue) in the presence of ADP/BeF3 is followed by fluorescence intensity measurements. Exponential fits to fluorescence time traces are shown in black and the unwinding rates obtained from the fits are given.
