The NS3 helicase from hepatitis C virus is a particularly valuable system for understanding the mechanism of helicase enzymes and their function in the context of large macromolecular machines. NS3 is active alone and in complex with other components of the replication machinery that modulate its behavior (Lindenbach, Fields Virology 2007). For example, activity by the NS3 helicase domain is regulated and enhanced by an appended viral protease domain. RNA binding by NS3 is also enhanced by interactions with the viral NS4A protein (Beran & Pyle, JBC 2007). NS3 is phylogenetically representative of the DExH group of helicases and it is structurally well-characterized (Yao & Webber, Structure 1998; Kim & Caron, Structure 1998).

To deduce the molecular mechanisms of RNA unwinding by NS3, the substrate specificity and the influence of partner proteins, we have utilized a diversity of approaches for monitoring helicase function that range from viral genetics to single molecule kinetic methodologies. NS3 displays many kinds of dynamic behaviors during the process of RNA unwinding, and each of these is crucial for the molecular mechanism. For example, single-molecule FRET and optical trapping experiments (conducted in collaboration with Taekjip Ha and Carlos Bustamante, respectively) indicate that the NS3 opens duplexes in abrupt, three-base pair steps that are coupled to ATP-dependent single-nucleotide translocation steps along the phosphodiester backbone (Myong et al, Science 2007; Dumont et al, Nature 2006). However, optical trap experiments and synchronized bulk measurements of helicase velocity indicate that NS3 takes a third type of large step that typically spans 9-18 base pairs, depending on the oligomeric state of the protein (Serebrov & Pyle, Nature 2004; Dumont et al, Nature 2006). These large steps are likely to represent the controlled binding and release of unwound product strands, which serves to reduce reannealing of the duplex. Taken together, studies of NS3 have provided the first high-resolution glimpses of helicase motion and highlighted the many parallels between nucleic acid motors and cytoskeletal motors such as kinesin (Pyle, Annu Rev Biophys 2008).