Poliovirus RNA-dependent RNA polymerase (3Dpol): structural, biochemical, and biological analysis of conserved structural motifs A and B.
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.
Gohara
D W
DW
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
Crotty
S
S
Arnold
J J
JJ
Yoder
J D
JD
Andino
R
R
Cameron
C E
CE
D000249:Adenosine Monophosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D054303:HIV Reverse Transcriptase / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017955:Poliovirus / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D008024:Ligands; D012367:RNA, Viral; D000249:Adenosine Monophosphate; D012324:RNA-Dependent RNA Polymerase; D054303:HIV Reverse Transcriptase