Poliovirus RNA-dependent RNA polymerase (3D(pol)): Structural, biochemical, and biological analysis of conserved structural motifs A and B

D. W. Gohara, S. Crotty, Jamie Jon Arnold, J. D. Yoder, R. Andino, Craig Eugene Cameron

Research output: Contribution to journalArticle

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Abstract

We have constructed a structural model for poliovirus RNA-dependent RNA polymerase (3D(pol)) in complex with a primer-template (sym/sub) and ATP. Residues found in conserved structural motifs A (Asp-238) and B (Asn-297) are involved in nucleotide selection. Asp-238 appears to couple binding of nucleotides with the correct sugar configuration to catalytic efficiency at the active site of the enzyme. Asn-297 is involved in selection of ribonucleoside triphosphates over 2'-dNTPs, a role mediated most likely via a hydrogen bond between the side chain of this residue and the 2'-OH of the ribonucleoside triphosphate. Substitutions at position 238 or 297 of 3D(pol) produced derivatives exhibiting a range of catalytic efficiencies when assayed in vitro for poly(rU) polymerase activity or sym/sub elongation activity. A direct correlation existed between activity on sym/sub and biological phenotypes; a 2.5-fold reduction in polymerase elongation rate produced virus with a temperature-sensitive growth phenotype. These data permit us to propose a detailed, structural model for nucleotide selection by 3D(pol), confirm the biological relevance of the sym/sub system, and provide additional evidence for kinetic coupling between RNA synthesis and subsequent steps in the virus life cycle.

Original languageEnglish (US)
Pages (from-to)25523-25532
Number of pages10
JournalJournal of Biological Chemistry
Volume275
Issue number33
DOIs
StatePublished - Aug 18 2000

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RNA Replicase
Poliovirus
Ribonucleosides
Nucleotides
Structural Models
Viruses
Elongation
Phenotype
Poly U
Life Cycle Stages
Sugars
Life cycle
Hydrogen
Catalytic Domain
Hydrogen bonds
Substitution reactions
Adenosine Triphosphate
RNA
Derivatives
Kinetics

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Gohara, D. W. ; Crotty, S. ; Arnold, Jamie Jon ; Yoder, J. D. ; Andino, R. ; Cameron, Craig Eugene. / Poliovirus RNA-dependent RNA polymerase (3D(pol)) : Structural, biochemical, and biological analysis of conserved structural motifs A and B. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 33. pp. 25523-25532.
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Poliovirus RNA-dependent RNA polymerase (3D(pol)) : Structural, biochemical, and biological analysis of conserved structural motifs A and B. / Gohara, D. W.; Crotty, S.; Arnold, Jamie Jon; Yoder, J. D.; Andino, R.; Cameron, Craig Eugene.

In: Journal of Biological Chemistry, Vol. 275, No. 33, 18.08.2000, p. 25523-25532.

Research output: Contribution to journalArticle

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AU - Gohara, D. W.

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AU - Arnold, Jamie Jon

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AU - Andino, R.

AU - Cameron, Craig Eugene

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