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^{1, 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 inchworm^{5, 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.

1. Biophysics Graduate Group,
2. Molecular and Cell Biology Department,
3. Chemistry Department,
4. Physics Department and Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA
5. Department of Molecular Biophysics and Biochemistry and Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA
6. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
7. *These authors contributed equally to this work

Correspondence to: Carlos Bustamante^1,2,3,4,6 Correspondence and requests for materials should be addressed to C.B. (Email: carlos@alice.berkeley.edu).

Received 23 June 2005 | Accepted 17 October 2005

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