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. 2021 Oct 7;10:e70968. doi: 10.7554/eLife.70968

Figure 1. SARS-CoV-2 polymerase is a fast and processive RNA polymerase complex.

(A) Schematic of the magnetic tweezers assay to monitor RNA synthesis by the SARS-CoV-2 polymerase complex. A magnetic bead is attached to a glass coverslip surface by a 1043 long ssRNA construct that experiences a constant force F (35 pN if not mentioned otherwise). The polymerase, formed by nsp7, nsp8, and nsp12, assembles at the 3′-end of the RNA strand annealed to the template. The subsequent conversion of the ssRNA template into dsRNA reduces the end-to-end extension of the tether, signaling replication activity. (B) SARS-CoV-2 polymerase activity traces acquired at either 25°C (gray) or 37°C (black), showing bursts of nucleotide addition interrupted by pauses. The inset is a zoom-in of the traces captured in the red square. (C) The dwell times collected from (B) are assembled into a distribution that is fitted using a stochastic pausing model (see Materials and methods; solid lines). The model includes four different probability distribution functions that describe the event that kinetically dominates the dwell time: uninterrupted 10 nucleotide additions (green), exponentially distributed Pause 1 and Pause 2 (blue and cyan, respectively), and the power-law distributed backtrack (red). (D) The dwell time distribution in (C) is described by the viral RdRp kinetic model (adapted from Dulin et al., 2017). Fast nucleotide addition is achieved by the nucleotide addition burst (NAB) pathway with the nucleotide addition rate extracted from (C). Pause 1 and Pause 2 are the kinetic signatures of the slow and very slow nucleotide addition (SNA and VSNA, respectively) pathways, the latter being likely related to nucleotide mismatch incorporation. (E) Total replication time and (F) product length of SARS-CoV-2 polymerase activity traces at either 25°C or 37°C. The median total replication time and the mean product length are indicated above the violin plots, and represented as thick horizontal lines. The error bars represent one standard deviation extracted from 1000 bootstraps. (G) Dwell time distributions of SARS-CoV-2 polymerase activity traces at 25°C (gray circles) and 37°C (black circles) extracted from (B), and their respective fit to the stochastic-pausing model (corresponding solid lines). (H) Nucleotide addition rate (green), Pause 1 (dark blue), and Pause 2 (cyan) exit rates at either 25°C or 37°C (solid and hatched bars, respectively) extracted from (G). The error bars in (C and G) represent one standard deviation extracted from 1000 bootstraps. The error bars in (H) are one standard deviation extracted from 100 bootstraps.

Figure 1.

Figure 1—figure supplement 1. Experimental conditions of SARS-CoV-2 polymerase high throughput magnetic tweezers experiments.

Figure 1—figure supplement 1.

(A) SDS-PAGE analysis of SARS CoV-2 replicase proteins. Shown is a 15% polyacrylamide gel with 2 µg of purified nsp7 (9 kDa), nsp8 (22 kDa), and nsp12 (110 kDa). Broad-range molecular weight markers (Mr) and corresponding molecular weights are indicated. Gel was stained with Coomassie. (B) Schematic of the RNA hairpin construct assembled from hybridizing and ligating single-stranded RNAs. (C) Force as a function of the extension of the RNA hairpin is presented in (A). Increasing and decreasing force ramp represented in gray and black, respectively. The red arrow indicates the extension at 35 pN, the force applied in the measurements unless specified. (D) Force as a function of the extension of the RNA hairpin after conversion into dsRNA. Increasing and decreasing force ramp represented in gray and black, respectively. (E) Typical field of view containing ~450 hairpin tethered magnetic beads in a high throughput magnetic tweezers assay.
Figure 1—figure supplement 1—source data 1. Source image for the SDS-PAGE gel in Figure 1—figure supplement 2A.
Figure 1—figure supplement 2. Selection of SARS-CoV-2 polymerase elongation traces and reproducibility.

Figure 1—figure supplement 2.

(A) SARS-CoV-2 polymerase activity traces acquired at 35 pN and 500 µM NTPs. Slow traces (<0.5% of the total number of events) are discarded from subsequent analysis. (B) Nucleotide addition rate (green), Pause 1 (dark blue), and Pause 2 (cyan) exit rates for triplicate experiment acquired at 25 pN and 500 µM NTP. (C) Probabilities to enter Pause 1 (dark blue), Pause 2 (cyan), and the backtrack (red) states for triplicate experiment acquired as in (B). (D) Probabilities to enter Pause 1 (dark blue), Pause 2 (cyan), and the backtrack (red) states at either 25°C (solid bars) or 37°C (hatched bars). Error bars in (E, F, D) are one standard deviation extracted from 100 bootstraps.