FIGURE 5.

Nucleotide incorporation catalyzed by NS5pol in the elongation complex. A, kinetics of GTP incorporation are shown. NS5pol (0.6 μm) and P₁₂/T₂₆ (0.5 μm) in the reaction buffer were incubated at 37 °C for 1 h. Aliquots of this mixture were rapidly mixed with GTP (0.8 mm after mixing), and then the reactions were quenched at various time intervals in a quench-flow instrument. The concentration of NS5pol and P₁₂/T₂₆ after mixing was 0.3 and 0.25 μm, respectively. The time course of 13-mer formation is shown. The data were fit to a two-exponential equation; the fast exponential had a rate of 13.2 ± 0.8 s⁻¹ and an amplitude of 0.083 ± 0.002 μm, and the slow exponential had a rate of 0.22 ± 0.01 s⁻¹ and an amplitude of 0.094 ± 0.002 μm. B, shown is primer extension with a single nucleotide or all four nucleotides. After incubation of NS5pol and P₁₂/T₂₆ (the template of each RNA has different template base for next incoming nucleotide, see Table 1) in the reaction buffer for 1 h at 37 °C, each preincubated sample was mixed with an equal volume of a solution containing single nucleotide at 0.5 mm or all four nucleotides (each nucleotide is 0.5 mm) and 0.1 mg/ml heparin in the reaction buffer. The reactions were quenched after 90 s and analyzed by PAGE.