Point mutations which drastically affect the polymerization activity of encephalomyocarditis virus RNA-dependent RNA polymerase correspond to the active site of Escherichia coli DNA polymerase I.
The inhibitor sensitivity and functional domains of recombinant encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) have been extensively analyzed. The inhibitor profiles of EMC virus 3Dpol and Escherichia coli DNA-dependent RNA polymerase are distinct, and experiments with substrate analogs indicate that EMC virus 3Dpol lacks reverse transcriptase activity. Twenty amino acid substitutions were engineered in EMC virus 3Dpol based on sequence alignments of viral RNA-dependent RNA polymerases that identified conserved amino acid residues within motifs. Ten out of 17 conservative substitutions within the four most conserved motifs reduced the RNA polymerase activity of the mutants to 0-6% of the activity of the wild-type enzyme, demonstrating the importance of these amino acids in the structure and/or function of EMC virus 3Dpol. Remarkably, 5 of the 10 mutations in EMC virus 3Dpol which had the most drastic effect on its RNA polymerase activity (D240E, S293T, N302Q, G332A, and D333E) were found to correspond to active site residues in E. coli DNA-dependent DNA polymerase I (Klenow). Our results reveal that a basic structural and functional framework is conserved in the most distantly related classes of nucleic acid polymerases and demonstrate the validity of modeling the active site of an RNA-dependent RNA polymerase on the known structure of a DNA polymerase.
Sankar
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Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Crescent.
Porter
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AG
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics* / Q000378:metabolism; D004686:Encephalomyocarditis virus / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012324:RNA-Dependent RNA Polymerase; D004256:DNA Polymerase I