Helicases use the energy derived from NTP hydrolysis to pry apart the hydrogen bonds between base pairs. These enzymes have been characterized in various ways, but most methods report only their bulk macro-scopic properties. The ability to follow helicase activity at the single-molecule level can provide insights into how the hydrolysis of NTP is directly coupled to translocation, unwinding of the duplex and processivity.

In a recent study, Dumont et al. (Nature 439, 105–108, 2006) use a single-molecule apparatus with high spatial and temporal resolution to examine the activities of hepatitis C virus NS3, an RNA helicase. Monomeric NS3 and ATP were placed in a chamber in which a 60-base-pair (bp) RNA hairpin with long single-strand arms was held by optical tweezers. Initially, NS3 binds the single-stranded region of the hairpin and translocates in the 3′→5′ direction. When NS3 encounters the duplex region and begins unwinding, the length of the RNA (reflected in the distance between the beads at the ends of the RNA) increases. Once the enzyme completes unwinding and dissociates, the hairpin re-forms, enabling multiple measurements from a single RNA molecule. In the illustration, NS3 is depicted as a workman climbing up a base-paired ladder, disrupting the steps as he moves.

Surprisingly, the unwinding traces are not monotonic but alternate between periods of rapid strand separation and pauses. This behavior is nonrandom; the rapid unwinding corresponds to a distance of 11 ± 3 bp, which is consistent with the enzyme's step size as obtained from previous bulk measurements (the pause is shown as a stop sign every 11 steps). In rare instances, backward movement over the same distance was observed. As individual unwinding events were recorded, information about the steps involved in unwinding could be extracted. For example, exit from a pause requires ATP binding, as expected, but it also has an ATP-independent component, the nature of which remains to be determined. The rate at which unwinding occurs between pauses (with vmax = 51 bp s−1) is dependent on ATP concentration. This result suggested that there are substeps within each 11-bp translocation, and careful examination of the tracings revealed the existence of 3.6-bp substeps, which has not been seen before. This observation is consistent with three cycles of ATP binding and hydrolysis per 11-bp unwinding event. Finally, processivity experiments indicated that NS3 is limited by its ability to translocate rather than its ability to separate strands and that reannealing competes with its translocation.

Overall, the kinetic properties determined by the single-molecule approach approximate the bulk measurements, with some of the differences attributed to the form (monomeric versus dimeric) of NS3 used. The authors propose a model for NS3 helicase activity, taking into account that there are two RNA-binding sites per monomer. In this model, one site is thought to contact the duplex 11 bp ahead of the enzyme after the paused enzyme binds ATP. The other (lagging) RNA-binding site is thought to act in duplex opening, translocating forward 3.6 bp during each of the three sequential ATP-binding and hydrolysis reactions. Other enzymes that disrupt base-pairing, such as polymerases, may also be studied using a variant of this assay. This may reveal some of the secrets of these molecular motors.