Noncatalytic Ions Direct the RNA-Dependent RNA Polymerase of Bacterial Double-Stranded RNA Virus 6 from De Novo Initiation to Elongation

RNA-dependent RNA polymerases (RdRps) are key to the replication of RNA viruses. A common divalent cation binding site, distinct from the positions of catalytic ions, has been identified in many viral RdRps. We have applied biochemical, biophysical, and structural approaches to show how the RdRp from bacteriophage6 uses the bound noncatalytic Mn2 to facilitate the dis-placement of the C-terminal domain during the transition from initiation to elongation.We find that this displacement releases the noncatalytic Mn2, which must be replaced for elongation to occur. By inserting a dysfunctional Mg2 at this site, we cap-tured two nucleoside triphosphates within the active site in the absence ofWatson-Crick base pairing with template andmapped movements of divalent cations during preinitiation. These structures refine the pathway from preinitiation through initiation to elongation for the RNA-dependent RNA polymerization reaction, explain the role of the noncatalytic divalent cation in6 RdRp, and pinpoint the previously unresolvedMn2-dependent step in replication. Viral RNA-dependent RNA polymerases (RdRps) share a sim-ilar structure and mechanism of catalysis and are potential targets for generic antiviral drugs. These enzymes resemble a closed right hand with fingers, palm, thumb, and fingertips do-mains (see Fig. 1A and Fig. 2A for domain structure). As in all known DNA and RNA polymerases, the nucleotidyltransferase reaction (35) involves two metal ions (Mg2) which are coordi