Limits...
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.

Show MeSH

Related in: MedlinePlus

Force dependence of the probability of entering into a backtracked state for P2 RdRp. (A) Probability density distributions for P2 RdRp transcriptional activity acquired at 16 pN (dark blue, 102 traces), 20 pN (light blue, 184 traces), 25 pN (green, 200 traces), 30 pN (yellow, 210 traces) and 35 pN (red, 76 traces). The error bars correspond to one standard deviation estimated from 1000 bootstraps. (B) A zoom-in of (A) for dwell-times longer than 10 s. (C) Probability that a dwell-time exceeds 20 s as a function of the applied force. The error bars are the standard deviation of the distribution extracted from 1000 bootstraps. (D) Proposed model that accounts for the force-dependence of the probability of finding P2 RdRp in a backtracked state. At low force (small distance between single nucleotides on the non-template strand), the tension at the dsRNA fork is small enough to allow the rehybridization of the template strand to the non-template strand, compensating the melting of the dsRNA product, leading to the backtracking of the RdRp. At high force (which results in a larger distance between single nucleotides on the non-template strand), the increase in tension impairs the rebridization of the template to the non-template strand, preventing backtracking to happen.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4666362&req=5

Figure 2: Force dependence of the probability of entering into a backtracked state for P2 RdRp. (A) Probability density distributions for P2 RdRp transcriptional activity acquired at 16 pN (dark blue, 102 traces), 20 pN (light blue, 184 traces), 25 pN (green, 200 traces), 30 pN (yellow, 210 traces) and 35 pN (red, 76 traces). The error bars correspond to one standard deviation estimated from 1000 bootstraps. (B) A zoom-in of (A) for dwell-times longer than 10 s. (C) Probability that a dwell-time exceeds 20 s as a function of the applied force. The error bars are the standard deviation of the distribution extracted from 1000 bootstraps. (D) Proposed model that accounts for the force-dependence of the probability of finding P2 RdRp in a backtracked state. At low force (small distance between single nucleotides on the non-template strand), the tension at the dsRNA fork is small enough to allow the rehybridization of the template strand to the non-template strand, compensating the melting of the dsRNA product, leading to the backtracking of the RdRp. At high force (which results in a larger distance between single nucleotides on the non-template strand), the increase in tension impairs the rebridization of the template to the non-template strand, preventing backtracking to happen.

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).


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)

Force dependence of the probability of entering into a backtracked state for P2 RdRp. (A) Probability density distributions for P2 RdRp transcriptional activity acquired at 16 pN (dark blue, 102 traces), 20 pN (light blue, 184 traces), 25 pN (green, 200 traces), 30 pN (yellow, 210 traces) and 35 pN (red, 76 traces). The error bars correspond to one standard deviation estimated from 1000 bootstraps. (B) A zoom-in of (A) for dwell-times longer than 10 s. (C) Probability that a dwell-time exceeds 20 s as a function of the applied force. The error bars are the standard deviation of the distribution extracted from 1000 bootstraps. (D) Proposed model that accounts for the force-dependence of the probability of finding P2 RdRp in a backtracked state. At low force (small distance between single nucleotides on the non-template strand), the tension at the dsRNA fork is small enough to allow the rehybridization of the template strand to the non-template strand, compensating the melting of the dsRNA product, leading to the backtracking of the RdRp. At high force (which results in a larger distance between single nucleotides on the non-template strand), the increase in tension impairs the rebridization of the template to the non-template strand, preventing backtracking to happen.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Force dependence of the probability of entering into a backtracked state for P2 RdRp. (A) Probability density distributions for P2 RdRp transcriptional activity acquired at 16 pN (dark blue, 102 traces), 20 pN (light blue, 184 traces), 25 pN (green, 200 traces), 30 pN (yellow, 210 traces) and 35 pN (red, 76 traces). The error bars correspond to one standard deviation estimated from 1000 bootstraps. (B) A zoom-in of (A) for dwell-times longer than 10 s. (C) Probability that a dwell-time exceeds 20 s as a function of the applied force. The error bars are the standard deviation of the distribution extracted from 1000 bootstraps. (D) Proposed model that accounts for the force-dependence of the probability of finding P2 RdRp in a backtracked state. At low force (small distance between single nucleotides on the non-template strand), the tension at the dsRNA fork is small enough to allow the rehybridization of the template strand to the non-template strand, compensating the melting of the dsRNA product, leading to the backtracking of the RdRp. At high force (which results in a larger distance between single nucleotides on the non-template strand), the increase in tension impairs the rebridization of the template to the non-template strand, preventing backtracking to happen.
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).

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