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A mechanism for initiating RNA-dependent RNA polymerization

Abstract

In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase. Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA. Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail1,2. Here we show that the 2 Å resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage φ6 (refs 3, 4) is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route. The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction. Our results provide a working model for the initiation of replication and transcription.

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Figure 1: Structure of φ6 polymerase and comparison with HCV polymerase.
Figure 2: Key aspects of various φ6 polymerase structures.
Figure 3: Models for initiation and chain elongation. a Cartoon illustrating key points in the reaction mechanism for φ6 polymerase.

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Acknowledgements

J. Diprose and G. Sutton helped with synchrotron data collection; J. Diprose and S. Ikemizu with calculations; and R. Esnouf and K. Harlos with computing and in-house data collection. We thank the staff at the beamlines of the ESRF, SRS and APS, in particular Sergey Korolev at the APS for help with the MAD experiment. S.J.B. is a Marie Curie Fellow. J.M.G. is funded by the Royal Society and D.I.S. by the Medical Research Council. The work was supported by the Academy of Finland, the Medical Research Council and the European Union.

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Correspondence to David I. Stuart.

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Butcher, S., Grimes, J., Makeyev, E. et al. A mechanism for initiating RNA-dependent RNA polymerization. Nature 410, 235–240 (2001). https://doi.org/10.1038/35065653

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