| Summary: | The biological role of manganese (MN^2+) 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 MN^2+ by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn^2+ -binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn^2+, reduced RNA binding and a compromised elongation rate. Loss of Mn^2+ 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: Mn^2+ 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 Mn^2+ is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.
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