Nucleotide binding by the poliovirus RNA polymerase.
Cross-linking of ribonucleoside triphosphates (NTPs) to specific binding sites on the poliovirus RNA-dependent RNA polymerase has been performed by ultraviolet irradiation and by reduction of oxidized nucleotide-protein complexes. The latter method approached a cross-linking efficiency of 1 NTP/molecule of enzyme. Nucleotide competition experiments suggested that the same binding site is occupied by all NTPs. Analysis of peptides produced by proteinase Glu-C and trypsin digestion and labeled with [32P]GTP indicated that a lysine residue between Met-189 and Lys-228 in the polymerase was cross-linked to NTP. Nucleotide binding was exploited for rapid purification of the enzyme by GTP-agarose affinity chromatography. In addition, a set of cloned, modified polymerase molecules with reduced or absent polymerization activity was analyzed for binding efficiency to a GTP-agarose column. Some mutations eliminated GTP binding, whereas others generated proteins with varying affinities for GTP. Incubation of the poliovirus polymerase with high concentrations of NTP, particularly GTP, resulted in a dramatic protection against heat denaturation and activity loss. These data suggest that nucleotide binding results in an alteration of the enzyme conformation or the stabilization of an ordered conformation.
Richards
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Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Yu
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Neufeld
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Ehrenfeld
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D001345:Autoradiography; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism*
D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D012265:Ribonucleotides; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases