ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding

Abstract

DEAD-box proteins, the largest helicase family, catalyze ATP-dependent remodeling of RNA-protein complexes and the unwinding of RNA duplexes. Because DEAD-box proteins hydrolyze ATP in an RNA-dependent fashion, the energy provided by ATP hydrolysis is commonly assumed to drive the energetically unfavorable duplex unwinding. Here, we show efficient unwinding of stable duplexes by several DEAD-box proteins in the presence of the nonhydrolyzable ATP analog ADP-beryllium fluoride. Another ATP analog, ADP-aluminum fluoride, does not promote unwinding. The findings show that the energy from ATP hydrolysis is dispensable for strand separation. ATP binding, however, appears necessary. ATP hydrolysis is found to be required for fast enzyme release from the RNA and multiple substrate turnovers and thus for enzyme recycling.

Fig. 1.

ADP-BeF_x promotes duplex unwinding by Ded1p. (A) Chemical structures of the terminal phosphate groups of ATP and the analogs used. (B) Unwinding reactions of 0.1 nM substrate (13 bp with 3′ 25-nt unpaired RNA) with 1.1 μM Ded1p in the absence or presence of 0.5 mM ATP or analog (0.5 mM ADP for ADP-BeF_x and ADP-AlF₄) as indicated. Reactions proceeded for 60 min. Diagrams indicate the RNA species; asterisks mark the radiolabel. (C) Unwinding reactions (60 min) with individual components of the noncovalent ADP-BeF_x. Components were present at identical concentrations in the experiments. (D) Binding of Ded1p to a 25-nt ssRNA (sequence of the unpaired RNA region of the substrate in Fig. 1A) under conditions identical to those in the unwinding reactions above. Components were incubated for 60 min. Before application to PAGE, excess ssRNA (1 μM final, 73-nt scavenger RNA; see Methods) was added and incubated for 1 min to bind free and remove loosely bound Ded1p from the substrate RNA. Diagrams indicate bound and free RNA.

Fig. 2.

Duplex unwinding with ADP-BeF_x and ATP display similar characteristics. (A) Unwinding reactions (60 min) of a 13-bp blunt-end substrate (0.1 nM) with 1.1 μM Ded1p in the absence or presence of 0.5 mM ATP (Left) or ADP-BeF_x (Right). (B and C) Dependence of unwinding rate constants on the Ded1p concentration with 0.5 mM ATP (B) and 0.5 mM ADP-BeF_x (C) for substrates with 13-bp (●) and 16-bp (○) and identical 25-nt unpaired RNA regions at the 3′ end. Asterisks indicate the radiolabels. Unwinding rate constants were determined as described (21). Error bars indicate the SD of 2 or more independent measurements; curves signify a trend through the data points.

Fig. 3.

ADP-BeF_x promotes duplex unwinding by different DEAD-box proteins, but not by a non-DEAD-box RNA helicase. Unwinding reactions of 0.1 nM substrate with 13-bp and 25-nt unpaired RNA at the 3′ end with 1 μM Mss116p (19 °C), 2 μM eIF4A (37 °C), and 100 nM NPH-II (19 °C) at 0.5 mM ATP and 0.5 mM ADP-BeF_x as indicated. Protein was present in all reactions. Reactions proceeded for 60 min. Diagrams indicate the RNA species; the asterisks mark the radiolabel.

Fig. 4.

ATP hydrolysis is necessary for fast enzyme release from RNA. (A) Simultaneous monitoring of duplex unwinding and Ded1p–RNA complex. Diagrams indicate the substrate and Ded1p. The DNA strand is gray, RNA is black, and Ded1p is depicted as semitransparent circle. The asterisk indicates the radiolabel on the RNA strand. DNA–RNA substrate (0.1 nM final) with 16-bp and 25 nt unpaired RNA at the 3′ end was incubated with 1.13 μM Ded1p for 10 min. The unwinding reaction was then started by addition of ATP or ADP-BeF_x (0.5 mM final) and allowed to proceed for 60 min. Subsequently, the reaction was divided in 2 parts. One part was terminated with SDS (see Methods) to remove the protein from the RNA; the other part was terminated with excess ssRNA (1 μM final, 73-nt scavenger RNA; see Methods) and EDTA. Both samples were applied to 2 different nondenaturing PAGE to visualize the unwound duplex (SDS) and the RNA protein complex (EMSA). (B) Nondenaturing PAGE to simultaneously monitor Ded1p–RNA complex (EMSA) and strand separation (SDS) during and unwinding reaction. Diagrams indicate the substrate species as described in A.

Fig. 5.

Turnover of excess substrate requires ATP hydrolysis. Representative reactions for unwinding of excess RNA substrate (1 μM, 13 bp with 25-nt single-stranded region at 3′; Fig. 1) by Ded1p (100 nM) with 0.5 mM ATP (Upper) and 0.5 mM ADP-BeF_x (Lower). Diagrams indicate the substrate species. The rate of substrate unwinding with ATP corresponds to v_obs = 0.125 μM·min⁻¹. After 20 min 2.5 μM substrate is unwound. With ADP-BeF_x, ≈0.05 μM substrate is unwound after 20 min.

Fig. 6.

The basic mechanism by which DEAD-box proteins couple ATP binding and hydrolysis to duplex unwinding. RNA strands are depicted as lines, and Ded1p is semitransparent circles. ATP and ADP are small squares. The single-stranded substrate region is gray, and the dotted line emphasizes that this region does not need to be physically connected to the RNA duplex to aid enzyme loading (6). The asterisk on the partially unwound RNA species without protein indicates the transient existence of this species. Unwinding reactions described here are started with the enzyme loading step. More than 2 Ded1p protomers may participate in the unwinding reaction (8). The mode of association of the 2 protomers to the RNA substrate is speculative.