Viral RNA helicases of the NS3/NPH-II group unwind RNA duplexes by processive, directional translocation on one of the duplex strands. The translocation is preceded by a poorly understood unwinding initiation phase. For NPH-II from vaccinia virus, unwinding initiation is rate limiting for the overall unwinding reaction. To develop a mechanistic understanding of the unwinding initiation, we studied kinetic and thermodynamic aspects of this reaction phase for NPH-II in vitro, using biochemical and single molecule fluorescence approaches. Our data show that NPH-II functions as a monomer and that different stages of the ATP hydrolysis cycle dictate distinct binding preferences of NPH-II for duplex versus single-stranded RNA. We further find that the NPH-II-RNA complex does not adopt a single conformation but rather at least two distinct conformations in each of the analyzed stages of ATP hydrolysis. These conformations interconvert with rate constants that depend on the stage of the ATP hydrolysis cycle. Our data establish a basic mechanistic framework for unwinding initiation by NPH-II and suggest that the various stages of the ATP hydrolysis cycle do not induce single, stage-specific conformations in the NPH-II-RNA complex but primarily control transitions between multiple states.
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