Figure 4.
The pH dependency of WT RdRP and its mutants in the elongation mode. (A–E) Using the T33-Fint/P10 construct, and following the same EC assembly and stopped-flow operation as in Figure 3, the single-nucleotide incorporation rates for the WT (A) and its mutants (B–E) were determined in a pH range of 4.5–11.0. Data from valid measurements are shown by filled squares, while open and filled circles shown at rate ‘0’ (indicated by the horizontal gray line) represent measurements not suitable for rate determination (see also Supplementary Table S1). Tris-derived WT data points (blue squares) were fitted to either a one-ionizable-group model or a two-ionizable-group model but with a constant pKa of the second ionization group (pKa,2: 10.5 or 12.0 for the WT), while only the one-ionizable-group model was used for all mutants. The WT curve generated using the one-ionizable-group model (red) in (A) is provided as a reference in (B–E).