Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site.
Bottom Line: We characterize the probability of entering long backtracks as a function of force and propose a model in which the bias toward backtracking is determined by the base paring at the dsRNA fork.We further discover that extensive backtracking provides access to a new 3'-end that allows for the de novo initiation of a second RdRp.This previously unidentified behavior provides a new mechanism for rapid RNA synthesis using coupled RdRps and hints at a possible regulatory pathway for gene expression during viral RNA transcription.
Affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.Show MeSH
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Mentions: To examine the force dependence of backtracking, we acquire data at forces of 16 pN, 20 pN, 25 pN, 30 pN and 35 pN (blue, cyan, green, yellow and red respectively, Figure 2A) using the buffer conditions described in Materials and Methods. In this range, the applied force destabilizes the ds-ssRNA junction by (8). We perform a dwell-time analysis for each force (Figure 2A,2B) and find that the recorded dwell-times span almost five orders of magnitude in time. Each distribution contains between ≈12000 (35 pN) and ≈30000 (20 pN) dwell-times. From Figure 2B, we observe that the long-lived pauses representing the backtracked state (Figure 1D) are populated to a degree that depends on the applied force: higher force provides lower probability density distribution of the dwell-times. We observe a force dependent hierarchy from 16 pN to 35 pN, with the error bars (one standard deviation confidence interval extracted from 1000 bootstrapped data sets) of the different distributions being well separated. The probability density distribution of the dwell-times does not depend on the presence of P2 RdRp in the reaction buffer, as shown in a previous study (Figure S5 of (8)). In the same study, we also observed that the lifetime of the exponentially distributed pauses (Figure 1D) does not exceed 17 s at [NTP]opt (8), and to avoid the influence of shorter non-backtracked pauses (region (ii) in Figure 1D), we similarly score only pauses longer than 20 s as in (8). In this way, we measure the probability of P2 to pause longer than 20 s within 10 nt incorporation for each force (Figure 2C). We observe a strong force dependence, where for example the probability of being in a pause longer than 20 s during a 10 nt incorporation cycle is 0.0228 ± 0.0012 at 16 pN and decreases nearly 20-fold to 0.0013 ± 0.0003 at 35 pN (Figure 2C).
Affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.