Fig. 4: Substrate preference of MERS-CoV ExoN complex.
From: Structural and catalytic diversity of coronavirus proofreading exoribonuclease
a Exonucleolytic digestion of RNA substrates bearing different 3′-end nucleotides by MERS-CoV ExoN WT. The reactions were stopped at indicated time points, and RNA products were resolved by denaturing PAGE and visualized by FAM (fluorescein) imaging. A representative result from three biological replicates is shown. b Percentages of substrate RNAs remaining shown in a were quantified using Bio-Rad Image Lab from three independent experiments and are shown as mean ± SEM. The results were plotted in GraphPad Prism using the One-phase decay model. Rate constant (k) values are indicated. Statistical analyses were performed using the two-sided extra sum-of-squares F test57. P values for the comparison of rate constants between T20P14-misC and each of the other four RNAs are indicated. c Fluorescence polarization analysis of the binding between MERS-CoV ExoN complex and different RNA substrates. Each data point represents the mean of six biological replicates ± SEM. Dissociation constant (KD) values are indicated. Statistical analyses were performed using the two-sided extra sum-of-squares F test. P values for the comparison of KD between T20P14-misC and each of the other four RNAs are indicated. d Exonucleolytic digestion of RNA substrates bearing different 3′-end nucleotides by MERS-CoV ExoN H95A mutant. A representative result from three biological replicates is shown. e Percentages of substrate RNAs remaining shown in d were quantified using Bio-Rad Image Lab from three independent experiments and are shown as mean ± SEM. The results were plotted in GraphPad Prism using the One-phase decay model. Rate constant (k) values are indicated. f Comparison of the rate constants of RNA digestion by MERS-CoV ExoN WT and H95A mutant. Data are shown as best-fit rate constant values ± 95% confidence interval (CI) of three biological replicates determined from curve fitting in b and e. Statistical analyses were performed using the two-sided extra sum-of-squares F test. P values are indicated. Source data are provided as a Source Data file.
