RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP

Abstract

Helicases are a ubiquitous class of enzymes involved in nearly all aspects of DNA and RNA metabolism. Despite recent progress in understanding their mechanism of action, limited resolution has left inaccessible the detailed mechanisms by which these enzymes couple the rearrangement of nucleic acid structures to the binding and hydrolysis of ATP. Observing individual mechanistic cycles of these motor proteins is central to understanding their cellular functions. Here we follow in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer. NS3 is a representative superfamily-2 helicase essential for viral replication, and therefore a potentially important drug target. We show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of 11 +/- 3 base pairs, and that actual unwinding occurs in rapid smaller substeps of 3.6 +/- 1.3 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm. This ATP-coupling mechanism is likely to be applicable to other non-hexameric helicases involved in many essential cellular functions. The assay developed here should be useful in investigating a broad range of nucleic acid translocation motors.

Figure 1. Assay with optical tweezers for assessing the mechanistic cycle of NS3

a, Experimental design and attachment of the RNA substrate. Not to scale. b, Stages of an unwinding experiment: the substrate is first unfolded and refolded with mechanical force (green), next brought to a constant force chosen between 5 and 17 pN to monitor NS3-catalysed unwinding (red), then brought to 30 pN to probe its state (blue), and finally brought to 2 pN to allow refolding (yellow; 50% of traces, as this one, display incomplete substrate refolding because of NS3 binding). c, Representative trace of extension against time unwinding (15 pN, from b). d, Pairwise distance distribution for the unwinding trace in c (1-bp bins).

Figure 2. [ATP] affects both NS3 pauses and steps

a, Representative traces of extension against time unwinding with 5 nM NS3 at 1 mM ATP (black), 0.1 mM ATP (red) and 0.05 mM ATP (blue). Traces are displaced along the time axis to avoid overlap. b, Histograms of pause durations (0.4-s bins) at 1 mM ATP (grey; 102 traces), 0.1 mM ATP (red; 52 traces) and 0.05 mM ATP (blue; 50 traces). c, Histograms of stepping velocities (5 bp s⁻¹ bins) for the data used in b. d, Stepping velocity as a function of [ATP] with Michaelis–Menten fit. Error bars show s.d.

Figure 3. NS3 steps are composed of substeps

Number of base pairs unwound over time for representative steps observed with 5 nM NS3 at 1 mM ATP (a), 0.1 mM ATP (b) and 0.05 mM ATP (c and d, displaying different numbers of substeps). Arrows point to substeps. About 90% of the steps observed at 1 mM ATP show no visible substeps within the step.

Figure 4. Effect of force on the behaviour of NS3, and proposed model of action

a, Fraction of duplex RNA of different lengths that are unwound processively at different forces. The mean processivity of NS3 is 18, 20, 33, 46, 48 and 53 bp at 5, 9, 11, 13, 15 and 17 pN (21, 69, 64, 62, 69 and 67 traces, respectively; 1-bp bins). b, Stepping velocities (red squares; means ± s.d.) and pause duration (blue circles; means and 95% fit confidence intervals) in 5 nM NS3 at 9, 11, 13, 15 and 17 pN (18, 35, 19, 53 and 39 traces, respectively). Pause durations plotted here were determined from a single-exponential fit of the pause duration histograms (Supplementary Fig. 4). c, Proposed model of NS3 translocation and unwinding. The helix opener site (red ellipse) unwinds the substrate in substeps of 2–5 bp (x and y designate substep sizes) triggered by ATP (V _max, K _m). The translocator (blue circle) contacts dsRNA every 11 bp, and possibly elsewhere during the cycle as well, which requires ATP binding (k _b) and an [ATP]-independent kinetic step (k _o).