FIGURE 7.

Biochemical characterization of the nascent RNA channel mutants. (A) Recombinant protein purification. The purified proteins were separated by SDS-PAGE (4%–12% polyacrylamide) and visualized by Coomassie Blue stain. (B) RNA synthesis using LE19 as the template RNA sequence. LE19 is capable of directing primer-dependent (32-nt product) and primer-independent (de novo) RNA synthesis (Ranjith-Kumar et al. 2001). Larger species are products of template-switch RNA synthesis. Quantification of de novo and primer extension RNA synthesis by 1bΔ21 NS5B and mutant proteins are below the gel. Averages and standard errors were from five independent experiments. (C) Affinities of RdRp and mutants to 12T as determined by fluorescence anisotropy experiments. 12T, at a final concentration of 0.2 μM, was resuspended in a buffer containing 50 mM HEPES (pH 7.5), 5 mM MgCl₂, 0.002% Tween 20, and 50 mM NaCl; RdRp or the nascent RNA channel mutant was titrated in. Sixty anisotropy values were taken after the RdRp was allowed to equilibrate for 1 min. The data were fitted into the Hill equation using KaleidaGraph software.