FIG. 3.

Activity of the purified EAV nsp9/RdRp on a poly(A)/oligo(dT) primer-template complex. (A) Time course of [³H]UTP incorporation (in cpm) on poly(A)/oligo(dT) using two concentrations of EAV RdRp (2 and 4 μM). As a specificity control, the reaction with 4 μM of enzyme was also carried out in the presence of rifampin (rif.), an inhibitor of E. coli DNA-dependent RNA polymerase. RNA synthesis was monitored in a filter-binding assay, followed by liquid scintillation counting. The data set on the right (C-) shows the results of a control reaction without the enzyme. (B) Optimization of the concentration of the catalytic ions Mg²⁺ and Mn²⁺ for RNA synthesis on poly(A)/oligo(dT). [³H]UTP incorporation (in cpm) after 30 min in the presence of increasing concentrations of Mg²⁺ and Mn²⁺ was measured by a filter-binding assay and liquid scintillation counting.