FIGURE 8.

RNA synthesis by the putative nascent RNA channel mutants in the presence of a nascent RNA mimic. (A) Change in fluorescence polarization while walking RdRps down 12T. Plotted is the change in fluorescence polarization under the conditions listed. All values are normalized to RdRp+12T. (B) Outline of the heparin titration experiment. RdRps were preincubated with either the 12T template alone or in the presence of the 5P primer. Heparin along with nucleotides were then added, and RNA synthesis was quantified by radiolabel incorporation. (C) The effect of heparin on RNA synthesis in the presence and absence of the 5P nascent RNA mimic used in the RCAP assay. Plotted is the relative abundance of RNA synthesized in the presence of different concentrations of heparin as indicated. In the presence of the 12T template alone, 1bΔ21 and the nascent RNA channel mutants showed discernable amounts of RNA synthesis (left panel). However, in the presence of 12T/5P, RNA synthesis by 1bΔ21 is significantly enhanced (right panel). This enhancement is not seen among any of the nascent RNA channel mutants.