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Nucleic Acids Res. 2008 Nov;36(20):6633-44. doi: 10.1093/nar/gkn632. Epub 2008 Oct 21.

Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase.

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1
Institute of Biotechnology and Department of Biological and Environmental Sciences, Viikki Biocenter, P.O. Box 56 (Viikinkaari 5) 00014 University of Helsinki, Finland.

Abstract

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 6 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.

PMID:
18940872
PMCID:
PMC2582606
DOI:
10.1093/nar/gkn632
[Indexed for MEDLINE]
Free PMC Article
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