Abstract
The NS3 helicase is essential for cytoplasmic RNA replication by the hepatitis C virus1,2,3,4, and it is a representative member of helicase superfamily 2 (SF2). NS3 is an important model system for understanding unwinding activities of DExH/D proteins5,6,7, and it has been the subject of extensive structural and mutational analyses8,9,10,11. Despite intense interest in NS3, the molecular and kinetic mechanisms for RNA unwinding by this helicase have remained obscure. We have developed a combinatorial, time-resolved approach for monitoring the microscopic behaviour of a helicase at each nucleotide of a duplex substrate. By applying this analysis to NS3, we have independently established the ‘physical’ and ‘kinetic’ step size for unwinding of RNA (18 base pairs, in each case), which we relate to the stoichiometry of the functional, translocating species. Having obtained microscopic unwinding rate constants at each position along the duplex, we demonstrate that NS3 unwinds RNA through a highly coordinated cycle of fast ripping and local pausing that occurs with regular spacing along the duplex substrate, much like the stepping behaviour of cytoskeletal motor proteins12.
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Acknowledgements
The authors would like to thank E. Jankowsky, J. Kawaoka and M. Brenowitz for discussions. We thank R. Beran for sharing his results on NS3 unwinding of multi-piece RNA substrates, and H. Le for discussions and early gifts of NS3 protein. This work was supported by grants from the NIH and the Howard Hughes Medical Institute. A.M.P. is an HHMI investigator.
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Supplementary information
Supplementary Figure 1
This figure describes NS3 titration experiments designed to determine functional oligomeric state of NS3: total NS3 binding to an RNA substrate as determined by filter binding assay is compared to functional binding (as monitored by the resulting unwinding amplitude). (PDF 13 kb)
Supplementary Figure 2
This figure shows model discrimination analysis of RNS unwinding. Timecourses for each RNS duplex length were fit to a simple sequential kinetic model (Scheme 1, Supplementary Methods), where the number of steps varied from 1 to 8. As shown on the figure, the squared residuals for each fit were plotted versus number of steps. The absolute minimum of squared residuals determines the number of rate-limiting steps for an RNS duplex of given length. (PDF 112 kb)
Supplementary Figure 3
Data from Supplementary Figure 2 are represented using color coding. Red color corresponds to the best fit for the total number of rate-limiting steps for each RNS duplex length. (PDF 8 kb)
Supplementary Figure 4
This figure shows four kinetic timecourses for NS3 unwinding of RNS duplexes within the same 18 bp kinetic step. The kinetic curves are virtually superimposable, although a small progressive deviation is observed for increasingly longer duplexes. (PDF 52 kb)
Supplementary Figure 5
This figure shows determination of physical step size and local processivity for an RNS duplex with a longer 3’-overhang (33 nt). The longer overhang does not change the pattern of local processivity along the duplex length. (PDF 86 kb)
Supplementary Methods
Supplementary Methods section includes extended description of kinetic analysis and global fitting for RNS unwinding by NS3. (DOC 35 kb)
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Serebrov, V., Pyle, A. Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature 430, 476–480 (2004). https://doi.org/10.1038/nature02704
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DOI: https://doi.org/10.1038/nature02704
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