Fig. 4.

Slow dsRNA binding amplifies the length dependence of MDA5. (A) Analysis of the RNA-binding kinetics of MDA5 using biotin pull-down. As with the dissociation assays in Fig. 3, the level of MDA5 bound to dsRNA was monitored using streptavidin magnetic beads and was quantitated by using the fluorescein tag on MDA5 (Methods and Materials). On the right is the time course of the bound fractions and fitted single exponential curves (mean ± SD, n = 3). (B) The binding rate constant (k_on) was obtained from the first-order approximation of the apparent binding rate (k_obs, obtained from Fig. S4B) against MDA5 concentration. (C) Time evolution of the ATP hydrolysis reaction initiated by the addition of ATP to the preformed complex of MDA5 (0.3 μM) and 112-bp dsRNA (0.6 μg/mL) (dsRNA→ATP) or by the addition of dsRNA to the preformed complex of MDA5 and ATP (ATP→dsRNA) (mean ± SD, n = 3). (D) Initial lag period of the ATP hydrolysis reactions of MDA5 (0.3 μM) using different concentrations of 112-bp dsRNA at 100 or 150 mM NaCl (mean ± SD, n = 3). The lag period was estimated from the linear extrapolation of the reaction time course as in C. (E) Relative ATP hydrolysis rates of MDA5 (0.3 μM) bound to model dsRNAs of 62- to 2,012 bp (4.8 μg/mL) at 100 or 150 mM NaCl (mean ± SD, n = 3). Rates are normalized against the rate measured with 2,012-bp dsRNA.