Figure 3. Impact of AMP on RNA helicase activity.

(a) Effect of adenosine nucleotides on duplex unwinding by Ded1p. Representative non-denaturing PAGE of unwinding time courses (30 min) with Ded1p.Cartoons show the mobility of unwound and duplex substrates, the asterisk marks the radiolabel. Unwinding reactions for Ded1p were performed as previously described ²⁴ (19 °C, 20 μl reaction buffer containing 40 mMTris·HCl (pH 8.0), 50 mMNaCl, 0.5 mM MgCl₂, 2 mM DTT, 0.6 unit•μl⁻¹RNasin, 0.01% (vol/vol) IGEPAL, 0.1 μM Ded1p, 0.5 nM 13 bp RNA duplex). Ded1p was pre-incubated with the 13 bp duplex substrate with a 25 nt 3’ss overhang (for sequences see Supplementary Materials) and the reactions were initiated with 0.5 mM ATP in the presence or absence of 0.5 mMindicated nucleotide (Total [Mg²⁺]: 1mM with ATP alone, 1.5 mM in all other reactions). At each time-point, a 3 μl portion was removed, and the reaction was stopped with 3 μl of 1% SDS, 50 mM EDTA, 20% (v/v) Glycerol, and 0.1% (w/v) Bromphenol Blue/Xylene Cylanol. Samples were applied to a 15% native polyacrylamide gel, and electrophoresed at 20 V/cm at 4°C. Gels were dried and radioactivity was quantified with a PhosphorImager (GE) and the ImageQuant software (Mol. Dynamics).First order unwinding rate constants were calculated as previously described ¹³.(b) Observed first order unwinding rate constants (k_unw) in the presence of indicated nucleotides, calculated for Ded1p from panel (a) and for several DEAD-box helicases (Mss116p, eIF4A, Sub2p, Dbp5p) and the non-DEAD-box RNA helicase Mtr4p. Error bars mark one standard deviation of at least three independent experiments. Unwinding reactions with the other RNA helicases were measured as described above, with the following modifications to obtain optimal unwinding rate constants: Mss116p: 10 mM Tris·HCl (pH 7.5), 100 mM KCl, Sub2p: 10 mM Tris·HCl (pH 7.0), 50 mM KCl at 30°C, Dbp5p: 40 mM Hepes (pH 7.5) at 30°C, eIF4A: 40 mM Hepes (pH 7.5), 8% (vol/vol) PEG6000 at 25°C and a 10bp duplex, and Mtr4p: 40 mM MOPS (pH 6.5), 100 mM NaCl, (16 bp duplex) at 30°C. Mtr4p was expressed and purified as described ^33,34. eIF4A was expressed and purified according to a protocol identical to that described for Ded1p ³³. Mss116p, Dbp5p, and Sub2p were generous gifts from Dr. Alan Lambowitz (University of Texas, Austin, TX), Dr. Karsten Weis (University of California, Berkeley, CA), and Dr. DitlevBroderson (University of Aarhus, Aarhus, Denmark). The proteins were expressed and purified as described ^26,35. Radiolabeled substrates were prepared as previously described ³⁶.