Figure 3.

SARS-CoV-2 RdRp Complex Weakly Binds an RNA Substrate and Dissociates at a Very Slow Rate

Scheme: ER_n is the enzyme-RNA complex with RNA n nucleotides in length k_off is the RNA dissociation rate and k_on is the apparent RNA association rate. The ratio of k_off/k_on gives the K_d or the apparent equilibrium binding constant. Kinetic constants for UTP incorporation are labeled as in the scheme in Figure 2. K_active is the equilibrium constant for enzyme going from the active E state to the inactive X state, which occurs during the pre-equilibration step to estimate the active enzyme concentration, as described in the Transparent Methods. Experiments in panels A–C are the same as in Figure 2.

(A) UTP incorporation, concentration dependence. Data for the 20, 21, and 22 nt RNA are shown in red, green, and blue, respectively.

(B) UTP incorporation, concentration dependence. The concentration of total product versus time is plotted for each UTP concentration.

(C) UTP incorporation at 20 μM UTP. RNA of different lengths is colored as in (A).

(D) UTP incorporation, enzyme titration. A mixture of 0.2–10 μM NSP12/7/8 complex, 20 μM NSP8, 200 nM FAM-20/40 RNA, and 5 mM Mg²⁺ was mixed with 150 μM UTP to start the reaction. The amount of total product formed was plotted versus time for each enzyme concentration and fit by simulation in KinTek Explorer.

(E) RNA dissociation rate experiment. A solution containing 1.25 μM NSP12/7/8 complex, 6 μM NSP8, 100 nM FAM-20/40 RNA, and 5 mM Mg²⁺ was allowed to equilibrate for 30 min, then mixed with 2 mg/mL heparin in the first mixing step using the RQF-3 rapid-quench flow instrument. After the designated first mixing time (shown in the figure), the reaction was mixed with 125 μM UTP (concentration after dilution) from the quench syringe, held in the exit line for 50 milliseconds, then the reaction was quenched by mixing with EDTA to a final concentration of 0.3 M in a collection tube. The time axis in the figure is the time allowed for RNA dissociation after mixing with heparin trap, before the addition of nucleotide. The best fit by simulation is shown as the solid line yielding an RNA dissociation rate of 0.013 ± 0.001 s^-1

(F) Confidence contours used to estimate fraction active enzyme. Data in panels A–E were fit globally while allowing the equilibrium constant, K_active to vary, along with k_on (which compensates for variable active enzyme concentration). The data were fit to extract the percent of active enzyme and the corresponding k_on values, as described in the Transparent Methods. All other rate constants were also allowed to vary, except k₁ and k₃ that were locked at 100 μM⁻¹s⁻¹. Data in the panel show that the enzyme is at least 60% active.

(G) Confidence contours to define k_on and k_off. Data in panels D and E were fit assuming 80% active enzyme with all other rate constants locked at their best-fit values. This analysis give k_on = 0.008 ± 0.001 μM⁻¹s⁻¹ and k_off = 0.013 ± 0.001 s⁻¹ and K_d for RNA binding of 1.8 ± 0.2 μM.

(H) Confidence contours for UTP binding and incorporation. Date in panels A–C were fit assuming 80% active enzyme and with k_on and k_off locked at their best fit values. The values for k₁ and k₃ were locked at 100 μM⁻¹s⁻¹. For each contour, the dashed line shows the χ² threshold. The smooth lines in each panel were derived from the global data fit. Rate constants derived from global data fitting, assuming 80% active enzyme, are given in Table 1.