Adenosine 5′-O-(3-thio)triphosphate (ATPγS) is a substrate for the nucleotide hydrolysis and RNA unwinding activities of eukaryotic translation initiation factor eIF4A

ATPγS•Mg and ATP•Mg catalyze eIF4A-dependent unwinding of RNA. (A) The duplex RNA substrate used in the unwinding assay. (B) Separation of radiolabeled single-stranded RNA product (ssRNA) from radiolabeled duplex RNA substrate (dsRNA) by nondenaturing gel electrophoresis. RNA was incubated for 5 min at 90°C (lane 1), or with 1 μM eIF4A (subsaturating) and 0.75 μM eIF4B (saturating) for indicated times in the absence (lanes 2–7) or presence (lanes 8–15) of 2 mM ATPγS•Mg. (C) Quantification of the reactions shown in B. Fraction RNA unwound versus time in reactions containing no nucleotide (□) or 2 mM ATPγS•Mg (•), yielding observed rate constants (k_obs,unw) of 6.0 × 10⁻⁴ and 5.6 × 10⁻² min⁻¹, respectively. (D) Dependence of unwinding rate (k_obs,unw) on the concentration of ATP•Mg (○) and ATPγS•Mg (•) in the presence of 1 μM eIF4A and 0.75 μM eIF4B. The curves represent fits to the Michaelis-Menten model, yielding K_1/2^ATP•Mg = 52 ± 8 μM, K_1/2^ATPγS•Mg = 96 ± 18 μM and maximum k_obs,unw values of 1.1 ± 0.2 min⁻¹ and 0.052 ± 0.08 min⁻¹, respectively.