Fig. 1.
ADP-BeFx 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-BeFx 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-BeFx. 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.