Functional oligomerization of poliovirus RNA-dependent RNA polymerase.
Using a hairpin primer/template RNA derived from sequences present at the 3' end of the poliovirus genome, we investigated the RNA-binding and elongation activities of highly purified poliovirus 3D polymerase. We found that surprisingly high polymerase concentrations were required for efficient template utilization. Binding of template RNAs appeared to be the primary determinant of efficient utilization because binding and elongation activities correlated closely. Using a three-filter binding assay, polymerase binding to RNA was found to be highly cooperative with respect to polymerase concentration. At pH 5.5, where binding was most cooperative, a Hill coefficient of 5 was obtained, indicating that several polymerase molecules interact to retain the 110-nt RNA in a filter-bound complex. Chemical crosslinking with glutaraldehyde demonstrated physical polymerase-polymerase interactions, supporting the cooperative binding data. We propose a model in which poliovirus 3D polymerase functions both as a catalytic polymerase and as a cooperative single-stranded RNA-binding protein during RNA-dependent RNA synthesis.
D001483:Base Sequence; D003432:Cross-Linking Reagents; D015870:Gene Expression; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D003432:Cross-Linking Reagents; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012324:RNA-Dependent RNA Polymerase