Figure 4.
Effect of inactivation of one or two helicase cores on RNA-stimulated ATPase activity, RNA binding and unwinding in the presence of the RBDs. (A) Hera constructs with two RBDs and two, one, or no functional core: Hera (two cores, two RBDs; black), Hera/Hera_K51Q (heterodimer, one functional core, two RBDs; dark purple), and Hera_K51Q (dimeric core: two cores, no RBD; purple). (B) RNA-dependent ATPase activity of 0.15 μM of Hera (two cores, black; filled circles; same data as in Fig. 2B) and 0.30 μM of Hera/Hera_K51Q (one functional core; dark purple, half-filled circles). 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 Fig. S7A for original data. (C) Fluorescence equilibrium titrations of 32mer with Hera (black, filled circles; same data as in Fig. 2C), Hera/Hera_K51Q (dark purple, half-filled circles), and Hera_K51Q (purple, open circles). Lines are fits according to a 1:1 binding model (see “Materials and methods” section). (D) Fluorescence equilibrium titrations of 32/9mer with Hera (black, filled circles; same data as in Fig. 2D), Hera/Hera_K51Q (dark purple, half-filled circles), and Hera_K51Q (purple, open circles). Lines are fits according to a 1:1 binding model (see “Materials and methods” section). (E) Concentration dependence of observed rate constants kobs for binding of Hera (black, filled circles; same data as in Fig. 2E), Hera/Hera_K51Q (dark purple, half-filled circles), and Hera_K51Q (purple, open circles) to 32mer RNA. Rate constants were obtained by describing stopped-flow traces (see Supplementary Fig. S7B) with single-exponential functions. (F) Concentration dependence of observed rate constants kobs for 32/9mer unwinding by Hera (black, filled circles; same data as in Fig. 2G) and Hera/Hera_K51Q (dark purple, half-filled circles). Rate constants were obtained by describing fluorescence traces (see Supplementary Fig. S7C) with single-exponential functions.

Effect of inactivation of one or two helicase cores on RNA-stimulated ATPase activity, RNA binding and unwinding in the presence of the RBDs. (A) Hera constructs with two RBDs and two, one, or no functional core: Hera (two cores, two RBDs; black), Hera/Hera_K51Q (heterodimer, one functional core, two RBDs; dark purple), and Hera_K51Q (dimeric core: two cores, no RBD; purple). (B) RNA-dependent ATPase activity of 0.15 μM of Hera (two cores, black; filled circles; same data as in Fig. 2B) and 0.30 μM of Hera/Hera_K51Q (one functional core; dark purple, half-filled circles). 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 Fig. S7A for original data. (C) Fluorescence equilibrium titrations of 32mer with Hera (black, filled circles; same data as in Fig. 2C), Hera/Hera_K51Q (dark purple, half-filled circles), and Hera_K51Q (purple, open circles). Lines are fits according to a 1:1 binding model (see “Materials and methods” section). (D) Fluorescence equilibrium titrations of 32/9mer with Hera (black, filled circles; same data as in Fig. 2D), Hera/Hera_K51Q (dark purple, half-filled circles), and Hera_K51Q (purple, open circles). Lines are fits according to a 1:1 binding model (see “Materials and methods” section). (E) Concentration dependence of observed rate constants kobs for binding of Hera (black, filled circles; same data as in Fig. 2E), Hera/Hera_K51Q (dark purple, half-filled circles), and Hera_K51Q (purple, open circles) to 32mer RNA. Rate constants were obtained by describing stopped-flow traces (see Supplementary Fig. S7B) with single-exponential functions. (F) Concentration dependence of observed rate constants kobs for 32/9mer unwinding by Hera (black, filled circles; same data as in Fig. 2G) and Hera/Hera_K51Q (dark purple, half-filled circles). Rate constants were obtained by describing fluorescence traces (see Supplementary Fig. S7C) with single-exponential functions.

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