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

Abstract

Whereas ATPγS is often considered a nonhydrolyzable substrate for ATPases, we present evidence that ATPγS is a good substrate for the RNA-stimulated nucleotide hydrolysis and RNA unwinding activities of eIF4A. In the presence of saturating single-stranded poly(U) RNA, eIF4A hydrolyzes ATPγS•Mg and ATP•Mg with similar steady-state parameters (K_M^NTP•Mg = 66 and 58 μM and k_cat = 1.0 and 0.97 min⁻¹, respectively). ATPγS•Mg also supports catalysis of RNA unwinding within 10-fold of the rate supported by ATP•Mg. The identical steady-state rate parameters, in comparison with the expected difference in the intrinsic rate of hydrolysis for ATP and ATPγS, suggest a nonchemical rate-limiting step for nucleotide hydrolysis. These results raise caution concerning the assumption that ATPγS is a nonhydrolyzable ATP analog and underscore the utility of thio-substituted NTPs as mechanistic probes.

Keywords

• ATPγS
• DEAD box protein
• DExD/H box protein
• RNA helicase
• ATPase
• unwindase
• thio-effect
• AMP-PNP, adenylyl imidodiphosphate
• ATPγS, adenosine 5′-O-(3-thio)triphosphate
• dsRNA, double-stranded (duplex) RNA
• eIF, eukaryotic translation initiation factor
• SF1, Helicase Super-family 1
• SF2, Helicase Super-family 2
• ssRNA, single-stranded RNA
• UTR, untranslated region