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

Nature volume 439, pages 105–108 (2006)Cite this article

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 ATP1,2. 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 replication3, and therefore a potentially important drug target4. 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 inchworm5,6. 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.

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Figure 1: Assay with optical tweezers for assessing the mechanistic cycle of NS3.
Figure 2: [ATP] affects both NS3 pauses and steps.
Figure 3: NS3 steps are composed of substeps.
Figure 4: Effect of force on the behaviour of NS3, and proposed model of action.

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Acknowledgements

We thank H. V. Le from the Schering-Plough Research Institute for the NS3 plasmid; S. B. Smith, P. T. X. Li, Y. R. Chemla and J.-C. Liao for discussions and technical help; T. M. Lohman for critical reading of the manuscript, and members of our laboratories for discussions and critical reading of the manuscript. This research was supported by CIHR and FQRNT doctoral fellowships (S.D.), an NIH postdoctoral fellowship (R.K.B.), NIH (I.T., A.M.P., C.B.), DOE (C.B.), and HHMI grants to investigators A.M.P. and C.B.

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Author notes

  1. Sophie Dumont and Wei Cheng: *These authors contributed equally to this work

Authors and Affiliations

  1. Biophysics Graduate Group,

    Sophie Dumont & Carlos Bustamante

  2. Molecular and Cell Biology Department,

    Wei Cheng & Carlos Bustamante

  3. Chemistry Department,

    Ignacio Tinoco Jr & Carlos Bustamante

  4. Physics Department and Howard Hughes Medical Institute, University of California, Berkeley, California, 94720, USA

    Carlos Bustamante

  5. Department of Molecular Biophysics and Biochemistry and Howard Hughes Medical Institute, Yale University, New Haven, Connecticut, 06520, USA

    Victor Serebrov, Rudolf K. Beran & Anna Marie Pyle

  6. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, 94720, USA

    Carlos Bustamante

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Correspondence to Carlos Bustamante.

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Supplementary information

Supplementary Figures

Eight figures showing a variety of NS3 behaviours (Supplementary Figure 1), that the 11 bp step size is intrinsic to NS3 (Supplementary Figures 2 and 3), that two kinetic events are required for NS3 pause exit (Supplementary Figure 4), that NS3 steps consist of rapid 2–5 bp substeps (Supplementary Figure 5), and providing part of the evidence that NS3 acts as a monomer in the present assay (Supplementary Figures 6, 7 and 8). (PDF 904 kb)

Supplementary Discussion

Discussions on the variability in the step size of NS3; the comparison between bulk and single molecule experiments on both theoretical and experimental grounds; the evidence for a single active NS3 monomer; two inchworm model orientations consistent with the data; the role of ATP in coordinating translocator and helix opener movement; thermodynamic considerations on coupling between ATP and duplex unwinding; confirming substrate pairing, geometry and attachment; instrument resolution; and the effects of NS3 binding to the substrate. (PDF 188 kb)

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Dumont, S., Cheng, W., Serebrov, V. et al. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439, 105–108 (2006). https://doi.org/10.1038/nature04331

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