Journal
NUCLEIC ACIDS RESEARCH
Volume 36, Issue 20, Pages 6633-6644Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkn632
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Funding
- Finnish Centre of Excellence Program [2006-2011]
- Human Frontier Science Program [RGP0320/2001-M]
- SPINE2-COMPLEXES [LSHG-CT-2006-031220]
- Helsinki Graduate School in Biotechnology and Molecular Biology
- Kuopio Naturalists' Society
- Royal Society
- Medical Research Council, UK
- Academy of Finland
- [1213992]
- [1213467]
- Medical Research Council [G0500365] Funding Source: researchfish
- MRC [G0500365] Funding Source: UKRI
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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 phi 6 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.
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