A mechanism for initiating RNA-dependent RNA polymerization.
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 detail. Here we show that the 2 A resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage straight phi6 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.
Institute of Biotechnology and Department of Biosciences, Viikki Biocenter, PO Box 56 (Viikinkaari 5), 00014 University of Helsinki, Finland.
S J
SJ
Butcher
J M
JM
Grimes
E V
EV
Makeyev
D H
DH
Bamford
D I
DI
Stuart
D017126:Bacteriophage phi 6 / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D012330:RNA, Double-Stranded / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011994:Recombinant Proteins; D008345:Manganese; D012194:RNA-Directed DNA Polymerase; D008274:Magnesium