Poliovirus RNA-Dependent RNA Polymerase (3Dpol): Kinetic, Thermodynamic, and Structural Analysis of Ribonucleotide Selection

David W. Gohara, Jamie Jon Arnold, Craig Eugene Cameron

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Abstract

We have performed a kinetic and thermodynamic analysis of 3Dpol 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 3Dpol/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.

Original languageEnglish (US)
Pages (from-to)5149-5158
Number of pages10
JournalBiochemistry
Volume43
Issue number18
DOIs
StatePublished - May 11 2004

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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