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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophage phi 6 / enzymology*
  • Binding Sites
  • Guanosine Triphosphate / chemistry
  • Manganese / chemistry*
  • Models, Molecular
  • Mutation
  • RNA / biosynthesis
  • RNA-Dependent RNA Polymerase / chemistry*
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism
  • Templates, Genetic
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism

Substances

  • Viral Proteins
  • Manganese
  • RNA
  • Guanosine Triphosphate
  • RNA-Dependent RNA Polymerase