Poliovirus RNA-dependent RNA polymerase (3Dpol): kinetic, thermodynamic, and structural analysis of ribonucleotide selection

Biochemistry. 2004 May 11;43(18):5149-58. doi: 10.1021/bi035429s.

Abstract

We have performed a kinetic and thermodynamic analysis of 3D(pol) derivatives containing substitutions in the ribose-binding pocket with ATP analogues containing correct and incorrect sugar configurations. We find that Asp-238, a residue in structural motif A that is conserved in all RNA-dependent RNA polymerases, is a key determinant of polymerase fidelity. Alterations in the position of the Asp-238 side chain destabilize the catalytically competent 3D(pol)-primer/template-NTP complex and reduce the efficiency of phosphoryl transfer. The reduction in phosphoryl transfer may be a reflection of increased mobility of other residues in motif A that are required for stabilizing the triphosphate moiety of the nucleotide substrate in the active conformation. We present a structural model to explain how Asp-238 functions to select nucleotides with a correct sugar configuration and a correct base. We propose that this mechanism is employed by all RNA-dependent RNA polymerases. We discuss the possibility that all nucleic acid polymerases with the canonical "palm"-based active site employ a similar mechanism to maximize fidelity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Substitution / genetics
  • Aspartic Acid / genetics
  • Binding Sites / genetics
  • Catalytic Domain / genetics
  • Conserved Sequence
  • Kinetics
  • Models, Molecular
  • Phosphorylation
  • Poliovirus / enzymology*
  • RNA Replicase / chemistry*
  • RNA Replicase / genetics
  • RNA Replicase / metabolism*
  • Ribonucleotides / chemistry*
  • Ribonucleotides / metabolism
  • Ribose / metabolism
  • Substrate Specificity
  • Templates, Genetic
  • Thermodynamics

Substances

  • Ribonucleotides
  • Aspartic Acid
  • Ribose
  • polymerase 3Dpol, poliovirus
  • RNA Replicase