Unwinding Initiation by the Viral RNA Helicase NPH-II

Abstract

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 vs. single stranded RNA. We further find that the NPH-II-RNA complex does not adopt a single but 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.

Sen1 has unique structural features grafted on the architecture of the Upf1‐like helicase family

“…Similarly to what has previously been observed in the structures of nucleotide‐bound Upf1 Hel (Chakrabarti et al , 2011), the adenine ring is sandwiched between an apolar surface of RecA1 and an aromatic residue (Tyr1655) that is present in the linker connecting RecA1 to RecA2 and is part of motif IIIa (Fairman‐Williams & Jankowsky, 2012). In addition, the conserved side chain of Gln1339 forms a bidentate hydrogen‐bond interaction with the N6 and N7 moieties of the adenine ring (Fig EV2A).…”

Division of Labor in an Oligomer of the DEAD-Box RNA Helicase Ded1p

“…Single-molecule FRET was measured in a home-built, total internal reflection (TIR) microscope setup (NIKON TE300 microscope, Pentamax Gen III CCD, PRINCETON INSTRUMENTS, 50 mW, frequency doubled solid-state laser, CRYSTALASER) (Fairman-Williams and Jankowsky, 2012; Yang et al, 2007b) RNA samples were immobilized in a flow-cell, coated with PEG/biotinylated PEG (Roy et al, 2008). Biotinylated RNA samples were immobilized via streptavidin (MOLECULAR PROBES).…”

Scaling of hysteresis loop of interacting polymers under a periodic force

“…For examples, DNA-B, a ring like hexameric helicase that pushes through the DNA like a wedge [8]. Williams and Jankowsy [9] showed that viral RNA helicase NPH-II that hops cyclically from the double stranded (ds) to the single stranded (ss) part of DNA and back during the ATP hydrolysis cycle. It is also proposed that pulling force resulting from ATP consumption is used by proteasomes and mitochondrial to unfold proteins [10][11][12].…”