FIG. 4.

De novo and primer-dependent initiation of RNA synthesis on homopolymeric templates. Standard RdRp assay mixtures using 1 μg of JFH1 (A) or J6 (B) RdRp were incubated for 1 hour with a poly(C) (de novo) or a poly(C)/oligo(G₁₂) (primer-dependent) template with 10 μCi [α-³²P]GTP and various concentrations of cold GTP, as shown on the x axis. Incorporation of radioactivity was quantified by TCA precipitation and liquid scintillation counting. After background subtraction, the picomoles of incorporated GMP per microgram of enzyme per hour were calculated based on the ratio of labeled to unlabeled GTP. (C) Difference in de novo initiation efficiencies between JFH1 and J6 RdRp at various GTP concentrations. The data represent the ratio of specific activities of JFH1 versus J6 polymerase on poly(C) templates, as shown in panels A and B at the indicated GTP concentrations.