Sam McBride-Wright, D.Phil.

Double-stranded RNA viruses encode a single protein species containing RNA-dependent RNA polymerase (RdRP) motifs. This protein is responsible for RNA transcription and replication. The architecture of viral RdRPs resembles that of a cupped right hand with fingers, palm and thumb domains. Those using de novo initiation have a flexible structural elaboration that constitutes the priming platform. Here we investigate the properties of the C-terminal priming domain of bacteriophage ϕ6 to get… Show more

Double-stranded RNA viruses encode a single protein species containing RNA-dependent RNA polymerase (RdRP) motifs. This protein is responsible for RNA transcription and replication. The architecture of viral RdRPs resembles that of a cupped right hand with fingers, palm and thumb domains. Those using de novo initiation have a flexible structural elaboration that constitutes the priming platform. Here we investigate the properties of the C-terminal priming domain of bacteriophage ϕ6 to get insights into the role of an extended loop connecting this domain to the main body of the polymerase. Proteolyzed ϕ6 RdRP that possesses a nick in the hinge region of this loop was better suited for de novo initiation. The clipped C-terminus remained associated with the main body of the polymerase via the anchor helix. The structurally flexible hinge region appeared to be involved in the control of priming platform movement. Moreover, we detected abortive initiation products for a bacteriophage RdRP. Show less

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 bacteriophage ϕ6 uses the bound non-catalytic Mn2+ to facilitate the displacement of the C-terminal domain during transition from the initiation to elongation. We find that this displacement… Show more

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 bacteriophage ϕ6 uses the bound non-catalytic Mn2+ to facilitate the displacement of the C-terminal domain during transition from the initiation to elongation. We find that this displacement releases the non-catalytic Mn2+, which must be replaced for elongation to occur. By inserting a dysfunctional Mg2+ at this site we captured two NTPs within the active site in the absence of Watson-Crick base pairing with template and mapped 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 non-catalytic divalent cation in ϕ6 RdRp and pinpoint the previously unresolved Mn2+-dependent step in replication. Show less

The biological role of manganese (Mn2+) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage ϕ6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn2+ by biochemical, biophysical and structural analyses of the… Show more

The biological role of manganese (Mn2+) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage ϕ6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn2+ by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn2+-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn2+, reduced RNA binding and a compromised elongation rate. Loss of Mn2+ binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn2+ coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn2+ is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization. Show less