Cooperation of the helicase core(s) with the RBD(s): ATPase activity, RNA binding and unwinding. (A) Hera constructs with different relative orientations of the helicase core and a single RBD. Hera (two functional cores, two RBDs; black), the cis-heterodimer (one core, 1 RBD in cis; green), the trans-heterodimer (one core, 1 RBD in trans; cyan), the cis-like heterodimer (Hera/Hera_1–419_K51Q: one functional core, one RBD in cis; dark green), and the trans-like heterodimer (Hera_1–419/Hera_K51Q: one functional core, one RBD in trans; dark blue). (B) RNA-dependent ATPase activity of 0.15 μM of Hera (black; same data set as in Fig. 2B), and 0.30 μM of the cis-heterodimer (green), the trans-heterodimer (cyan), the cis-like heterodimer (dark green), and the trans-like heterodimer (dark blue). 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). See Supplementary Figs S1A and S10A for original data. (C) Fluorescence equilibrium titrations of 32mer with Hera (black; same data set as in Fig. 2C), the cis-heterodimer (green), the trans-heterodimer (cyan), and the RBD (blue). Lines are fits according to a 1:1 binding model (see “Materials and methods” section). (D) Fluorescence equilibrium titrations of double-stranded 32/9mer RNA with Hera (black; same data set as in Fig. 2D), the cis-heterodimer (green), the trans-heterodimer (cyan), and the RBD (blue). Lines are fits according to a 1:1 binding model (see “Materials and methods” section). Hera concentrations are given as concentrations of dimer. (E) Concentration dependence of observed rate constants k_obs for binding of Hera (black; same data set as in Fig. 2E), the cis-heterodimer (green), the trans-heterodimer (cyan), and the RBD (blue) to 32mer RNA. Rate constants were obtained by describing stopped-flow traces (see Supplementary Fig. S10B) with single-exponential functions. (F) Concentration dependence of observed rate constants k_obs for binding of Hera (black) and the cis-heterodimer (green) to 32/9mer RNA. Rate constants were obtained by describing stopped-flow traces with single-exponential functions. (G) Concentration dependence of observed rate constants k_obs for 32/9mer unwinding by Hera (black; same data as in Fig. 2F), the cis-heterodimer (green), the trans-heterodimer (cyan), the cis-like heterodimer (dark green), and the trans-like heterodimer (dark blue). Rate constants were obtained by describing the fluorescence traces with single-exponential functions.