Figure 4. T-1106-TP incorporation induces pauses of intermediate duration and backtrack.

(A) SARS-CoV-2 polymerase activity traces in the presence of 500 µM NTPs, in the presence of 500 µM T-1106-TP. The inset is a zoom-in of the polymerase activity traces captured in the black square. (B) SARS-CoV-2 replication time for the 1043 nt long template using 500 µM of all NTPs, and the indicated concentration of T-1106-TP. The median values are indicated above the violin plots, and represented by horizontal black thick lines flanked by one standard deviation error bars extracted from 1000 bootstraps. (C) SARS-CoV-2 polymerase product length using 500 µM NTPs and the indicated concentration of T-1106-TP. The mean values are indicated above the violin plots, and represented by horizontal black thick lines flanked by one standard deviation error bars extracted from 1000 bootstraps. (D) Dwell time distributions of SARS-CoV-2 polymerase activity traces for 500 µM NTP either without (gray) or with 500 µM (red) T-1106-TP. The corresponding solid lines are the fit to the stochastic-pausing model. (E) Nucleotide addition rate (green), Pause 1 (dark blue), and Pause 2 (cyan) exit rates for [NTPs]=500 µM and several T-1106-TP concentrations. (F) Probabilities to enter Pause 1 (dark blue), Pause 2 (cyan), and the backtrack (red) states for the conditions described in (E). The error bars denote one standard deviation from 1000 bootstraps in (D) and the error bars in (E, F) denote one standard deviation extracted from 100 bootstrap procedures.

Figure 4—figure supplement 1. SARS-CoV-2 polymerase elongation in presence of T-1106-TP.

Using an ultra-stable magnetic tweezers configuration, we monitored pauses in SARS-CoV-2 polymerase activity traces at 58 Hz camera acquisition frequency (gray; 1 Hz low-pass filtered: dark gray), applying 35 pN force, and in the presence of 500 µM T-1106-TP and 500 µM all NTPs. Top: zoom-in the polymerase activity traces; bottom inactive tether followed simultaneously. (A) Some pauses demonstrate shallow backtracks, (B) while in other pause cases, polymerase backtrack is difficult to confirm given the spatiotemporal resolution of the assay. (C) Dwell time distributions of SARS-CoV-2 polymerase activity traces acquired in the presence of 500 µM NTP, either without (circles), or with 20 µM (triangles), 50 µM (squares), 100 µM (diamonds), 300 µM (pentagons), and 500 µM (upside down triangle) T-1106-TP. The color code from light to dark gray further highlights the increasing concentration of T-1106-TP. The solid lines represent the fit of the stochastic-pausing model. The error bars represent one standard deviation extracted from 1000 bootstraps.