Poliovirus RNA-dependent RNA polymerase (3Dpol): kinetic, thermodynamic, and structural analysis of ribonucleotide selection.
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.
Gohara
David W
DW
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Arnold
Jamie J
JJ
Cameron
Craig E
CE
D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D007700:Kinetics; D008958:Models, Molecular; D010766:Phosphorylation; D017955:Poliovirus / Q000201:enzymology*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012265:Ribonucleotides / Q000737:chemistry* / Q000378:metabolism; D012266:Ribose / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D013816:Thermodynamics
D012265:Ribonucleotides; D001224:Aspartic Acid; D012266:Ribose; C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase