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Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site.

Dulin D, Vilfan ID, Berghuis BA, Poranen MM, Depken M, Dekker NH - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.

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Long pauses in the elongation dynamics are rarely followed by processive changes in the direction of transcription. (A) The extension of the transcribed product as a function of time. At 800 s (indicated by arrow), P2 RdRp exhibits a reversal behavior. The experimental conditions include an applied force of 16 pN force and an acquisition frequency of 25 Hz. (B) The probability of observing a reversal event as a function of the applied force (see panel (A)). The error bars represent the 95% confidence interval for a binomial distribution for the ordinate and the standard deviation of the applied force (±5%, see (50)) for the abscissa. (C and D) P2 RdRp transcription activity traces that exhibit very rare behavior in which multiple switches in the apparent directionality of the signal can be observed. In both panels, the extension of the RNA construct (1) increases, then (2) decreases (as in a reversal event) and (3) increases again. Data are acquired at an applied force of 25 pN using buffer conditions described in Materials and Methods. For panels (A), (C) and (D), raw data are shown in blue and data low-pass filtered at 0.5 Hz are shown in black.
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Figure 3: Long pauses in the elongation dynamics are rarely followed by processive changes in the direction of transcription. (A) The extension of the transcribed product as a function of time. At 800 s (indicated by arrow), P2 RdRp exhibits a reversal behavior. The experimental conditions include an applied force of 16 pN force and an acquisition frequency of 25 Hz. (B) The probability of observing a reversal event as a function of the applied force (see panel (A)). The error bars represent the 95% confidence interval for a binomial distribution for the ordinate and the standard deviation of the applied force (±5%, see (50)) for the abscissa. (C and D) P2 RdRp transcription activity traces that exhibit very rare behavior in which multiple switches in the apparent directionality of the signal can be observed. In both panels, the extension of the RNA construct (1) increases, then (2) decreases (as in a reversal event) and (3) increases again. Data are acquired at an applied force of 25 pN using buffer conditions described in Materials and Methods. For panels (A), (C) and (D), raw data are shown in blue and data low-pass filtered at 0.5 Hz are shown in black.

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).


Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site.

Dulin D, Vilfan ID, Berghuis BA, Poranen MM, Depken M, Dekker NH - Nucleic Acids Res. (2015)

Long pauses in the elongation dynamics are rarely followed by processive changes in the direction of transcription. (A) The extension of the transcribed product as a function of time. At 800 s (indicated by arrow), P2 RdRp exhibits a reversal behavior. The experimental conditions include an applied force of 16 pN force and an acquisition frequency of 25 Hz. (B) The probability of observing a reversal event as a function of the applied force (see panel (A)). The error bars represent the 95% confidence interval for a binomial distribution for the ordinate and the standard deviation of the applied force (±5%, see (50)) for the abscissa. (C and D) P2 RdRp transcription activity traces that exhibit very rare behavior in which multiple switches in the apparent directionality of the signal can be observed. In both panels, the extension of the RNA construct (1) increases, then (2) decreases (as in a reversal event) and (3) increases again. Data are acquired at an applied force of 25 pN using buffer conditions described in Materials and Methods. For panels (A), (C) and (D), raw data are shown in blue and data low-pass filtered at 0.5 Hz are shown in black.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4666362&req=5

Figure 3: Long pauses in the elongation dynamics are rarely followed by processive changes in the direction of transcription. (A) The extension of the transcribed product as a function of time. At 800 s (indicated by arrow), P2 RdRp exhibits a reversal behavior. The experimental conditions include an applied force of 16 pN force and an acquisition frequency of 25 Hz. (B) The probability of observing a reversal event as a function of the applied force (see panel (A)). The error bars represent the 95% confidence interval for a binomial distribution for the ordinate and the standard deviation of the applied force (±5%, see (50)) for the abscissa. (C and D) P2 RdRp transcription activity traces that exhibit very rare behavior in which multiple switches in the apparent directionality of the signal can be observed. In both panels, the extension of the RNA construct (1) increases, then (2) decreases (as in a reversal event) and (3) increases again. Data are acquired at an applied force of 25 pN using buffer conditions described in Materials and Methods. For panels (A), (C) and (D), raw data are shown in blue and data low-pass filtered at 0.5 Hz are shown in black.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.

Show MeSH
Related in: MedlinePlus