Effect of the number of helicase cores on RNA-stimulated ATPase activity, RNA binding and unwinding, and conformational changes of the helicase core in the absence of the RBDs. (A) Hera (two cores, two RBDs; black), Hera_1–419 (dimeric core: two cores, no RBD; red), and Hera_1–365 (monomeric core: one core, no RBD; orange). (B) RNA-dependent ATPase activity of 0.15 μM of Hera (black; same data as in Fig. 2B), 0.15 μM of Hera_1–419 (red, same data as in Fig. 2B), and 0.3 μM of Hera_1–365 (orange). Data are cumulative data points from at least two independent experiments. The lines are cumulative fits to all data points with the Michaelis–Menten equation (see “Materials and methods” section). (C) Fluorescence equilibrium titrations of 32mer with Hera (black; same data as in Fig. 2C), Hera_1–419 (red; same data as in Fig. 2C), and Hera_1–365 (orange). Lines are fits according to a 1:1 binding model (see “Materials and methods” section). (D) Concentration dependence of observed rate constants k_obs for 32/9mer unwinding by Hera (black; same data as in Fig. 2G), Hera_1–419 (red; same data as in Fig. 2G), and Hera1-365 (orange). Rate constants were obtained by describing fluorescence traces with single-exponential functions. (E) Single-molecule FRET histograms for donor–acceptor-labeled Hera (black; same data as in Fig. 3A), Hera_1–419 (red; same data as in Fig. 3A), and Hera_1–365 (orange) in the absence (lines, no fill) and presence of 5 mM ADPNP and 32mer RNA (4.8 μM with Hera, Hera_1–356, 3.2 μM with Hera_1–419; lines, colored fill). Representative histograms from at least two independent experiments. (F) FRET efficiency as a function of the concentration of 32mer RNA for donor–acceptor-labeled Hera (black; same data as in Fig. 3B), Hera_1–419 (red; same data as in Fig. 3B), and Hera_1–365 (orange) in the presence of 5 mM ADPNP. Error bars reflect the error of the mean from at least two independent experiments.