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: In our single-molecule experiments, we occasionally observe events in which P2 appears to ‘reverse’ its direction during transcription (Figure 3A). Prior to such events, P2 transcript elongation starts normally (Figure 3A, data up to ≈600 s). However, following a long pause, we occasionally observe a decrease in the extension of the RNA tether (Figure 3A, black arrow). This decrease typically continues until the tether length of the RNA construct reverts to its original value measured prior to P2 initiation. Such ‘reversal’ events occur in a small percentage of all traces. The occurrence of reversals in a trace is, however, significantly more likely at applied forces below 20 pN (Figure 3B), with a probability of 0.15 ± 0.07 and 0.027 ± 0.026 at applied forces of 16 pN and 35 pN, respectively. In very rare instances (0.2% of P2 traces collected), we also observe a renewed ‘forward’ motion upon the completion of a reversal (Figure 3C,3D).
Affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.