Figure 2. Connection between oligomerization, ATPase and helicase activity.

(A) Unwinding of two RNA substrates (2 µM) with differing duplex length under steady-state conditions with increasing concentrations of Ded1p. Data points here and in all subsequent figures are averages from multiple independent measurements (N ≥ 3) and error bars represent one standard deviation, unless indicated otherwise. Dashed lines indicate the dependence of the unwinding rate expected for a monomeric enzyme. Solid lines mark a trend.

(B) Unwinding of a 16 bp RNA substrate (0.5 nM) under pre-steady state conditions with increasing concentrations of Ded1p and Ded1p^ΔC. Solid lines are the best fits to the Hill equation (for wt Ded1p:k_unw^max = 7.4 ± 0.2 min⁻¹,K_1/2 = 320 ± 40 nM, H = 3.2 ± 0.1; for Ded1p^ΔC: no meaningful datafit)

(C) Inhibition of unwinding by Ded1p, but not Ded1p^ΔC with the C-terminal domain of Ded1p. Measurements were performed with 0.5 nM RNA (13bp, 25 nt 3’ ssRNA), 300 nM Ded1p,900 nM Ded1p^ΔC, 0.4 mM ATP, 1.0 µM GST-Ded1p-Ct or GST.

(D) RNA-stimulated ATPase activity with 50 nM RNA (16 bp, 25 nt 3’ ssRNA) at indicated concentrations of Ded1p and Ded1p^ΔC. Solid lines are the best fits to the Hill equation (for wt Ded1p:V_max = 2.1 ± 0.2 µM·min⁻¹, K_1/2 = 220 ± 60 nM, H = 1.6 ± 0.2; for Ded1p^ΔC: no meaningful datafit)

(E) Inhibition of helicase and RNA-stimulated ATPase activities of Ded1p with Ded1p^DAAD, measured at 0.5 nM (unwinding) or 50 nM (ATPase) RNA (16 bp duplex with 3' 25 ntssRNA), 1 µM total protein concentration ([Ded1p]+ [Ded1p^DAAD]), and 2 mM ATP. Activities are plotted as a fraction of the activity of 1 µM wtDed1p. The solid line indicates the expected result for a monomeric enzyme. Dashed mark a trend.

(F) Comparison of unwinding and ATPase activity of wtDed1p for a series of RNA-DNA hybrid substrates (cartoons on the left - grey broken line: DNA; black solid line: RNA). Values for k_cat/K_m for ATP hydrolysis were obtained with 2 µM nucleic acid substrate, 0.4 µM Ded1p, as in Fig. 3A, values for k_unw^max for unwinding with 0.1 nM substrate, 2 mM ATP/Mg²⁺, as in Fig. 2B. See also Figure S2.