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Reinitiated viral RNA-dependent RNA polymerase resumes replication at a reduced rate.

Vilfan ID, Candelli A, Hage S, Aalto AP, Poranen MM, Bamford DH, Dekker NH - Nucleic Acids Res. (2008)

Bottom Line: Here, we report that RdRP experience a dramatic, long-lived decrease in its elongation rate when it is reinitiated following stalling.The rate decrease has an intriguingly weak temperature dependence, is independent of both the nucleotide concentration during stalling and the length of the RNA transcribed prior to stalling; however it is sensitive to RNA structure.This allows us to delineate the potential factors underlying this irreversible conversion of the elongation complex to a less active mode.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Applied Sciences, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.

ABSTRACT
RNA-dependent RNA polymerases (RdRP) form an important class of enzymes that is responsible for genome replication and transcription in RNA viruses and involved in the regulation of RNA interference in plants and fungi. The RdRP kinetics have been extensively studied, but pausing, an important regulatory mechanism for RNA polymerases that has also been implicated in RNA recombination, has not been considered. Here, we report that RdRP experience a dramatic, long-lived decrease in its elongation rate when it is reinitiated following stalling. The rate decrease has an intriguingly weak temperature dependence, is independent of both the nucleotide concentration during stalling and the length of the RNA transcribed prior to stalling; however it is sensitive to RNA structure. This allows us to delineate the potential factors underlying this irreversible conversion of the elongation complex to a less active mode.

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Related in: MedlinePlus

A reinitiated φ6 RdRP elongation complex (φ6EC) and randomly-initiated φ6 RdRP replication show distinct electrophoretic profiles on agarose gels. (A) Schematic of stalling and reinitiation of φ6EC. In the presence of three NTPs (ATP, GTP, CTP), a φ6EC is stalled at the 50th nt from the 3′-end of the template at temperature T1. The sequence elongated prior to stalling is shown in blue. After UTP addition, the stalled φ6EC reinitiates and synthesizes the complementary strand at temperature T2. (B) Agarose gel of the elongation intermediates after reinitiation of the stalled φ6EC on 4 kb ssRNA template. Aliquots were taken at different times after reinitiation (telong). Letters S and P indicate 4 kb ssRNA and 4 kb dsRNA, respectively. (C) Schematic of a randomly-initiated φ6 RdRP replication. 4 kb ssRNA was incubated with φ6 RdRP, and all four NTPs were subsequently added simultaneously. (D) Agarose gel of aliquots of randomly-initiated φ6 RdRP replication taken at different polymerization times (tpoly).
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Figure 2: A reinitiated φ6 RdRP elongation complex (φ6EC) and randomly-initiated φ6 RdRP replication show distinct electrophoretic profiles on agarose gels. (A) Schematic of stalling and reinitiation of φ6EC. In the presence of three NTPs (ATP, GTP, CTP), a φ6EC is stalled at the 50th nt from the 3′-end of the template at temperature T1. The sequence elongated prior to stalling is shown in blue. After UTP addition, the stalled φ6EC reinitiates and synthesizes the complementary strand at temperature T2. (B) Agarose gel of the elongation intermediates after reinitiation of the stalled φ6EC on 4 kb ssRNA template. Aliquots were taken at different times after reinitiation (telong). Letters S and P indicate 4 kb ssRNA and 4 kb dsRNA, respectively. (C) Schematic of a randomly-initiated φ6 RdRP replication. 4 kb ssRNA was incubated with φ6 RdRP, and all four NTPs were subsequently added simultaneously. (D) Agarose gel of aliquots of randomly-initiated φ6 RdRP replication taken at different polymerization times (tpoly).

Mentions: In replication, a φ6 RdRP elongation complex (φ6EC) can be stalled in vitro using a limited selection of NTPs and a template molecule in which the 3′-terminal region is devoid of one or more of the nucleotides (Figure 2A; for proof of stalling on short oligos, see Supplementary Data and Figure S3). Elongation can then be reinitiated by the addition of the missing NTP(s), yielding an entirely double-stranded product. To study the effect of stalling on the rate of φ6 RdRP replication, we selected a 4193 nt long replication template (4 kb ssRNA) in which the first occurrence of adenine was 50 nt from the 3′-end. The stalled φ6EC exhibited an electrophoretic mobility indistinguishable from that of free 4 kb ssRNA (Supplementary Figure S4). Following stalling and reinitiation by UTP addition, aliquots were collected at successive time points and analyzed on agarose gel (Figure 2B). After reinitiation, a fraction of the replication template retained the electrophoretic mobility of free 4 kb ssRNA (Supplementary Figure 2B, lanes 2–10), which corresponds to either free 4 kb ssRNA or to inactive stalled φ6EC. The electrophoretic mobility of successfully reinitiated φ6ECs decreased with time as φ6 RdRP progressed along the 4 kb ssRNA (Figure 2B, lanes 2–10). Notably, the band corresponding to the reinitiated φ6ECs stayed well-defined, suggesting that the stalled φ6ECs reinitiated in a synchronized manner. Termination of the replication reaction was detected by a stabilization of the electrophoretic mobility of the reinitiated φ6ECs (data not shown). This occurred between 30 min and 60 min after reinitiation, from which we deduce an overall kelong between 1.2 and 2.3 nt·s−1. (For the discussion of the measured rates please see Supplementary Data).Figure 2.


Reinitiated viral RNA-dependent RNA polymerase resumes replication at a reduced rate.

Vilfan ID, Candelli A, Hage S, Aalto AP, Poranen MM, Bamford DH, Dekker NH - Nucleic Acids Res. (2008)

A reinitiated φ6 RdRP elongation complex (φ6EC) and randomly-initiated φ6 RdRP replication show distinct electrophoretic profiles on agarose gels. (A) Schematic of stalling and reinitiation of φ6EC. In the presence of three NTPs (ATP, GTP, CTP), a φ6EC is stalled at the 50th nt from the 3′-end of the template at temperature T1. The sequence elongated prior to stalling is shown in blue. After UTP addition, the stalled φ6EC reinitiates and synthesizes the complementary strand at temperature T2. (B) Agarose gel of the elongation intermediates after reinitiation of the stalled φ6EC on 4 kb ssRNA template. Aliquots were taken at different times after reinitiation (telong). Letters S and P indicate 4 kb ssRNA and 4 kb dsRNA, respectively. (C) Schematic of a randomly-initiated φ6 RdRP replication. 4 kb ssRNA was incubated with φ6 RdRP, and all four NTPs were subsequently added simultaneously. (D) Agarose gel of aliquots of randomly-initiated φ6 RdRP replication taken at different polymerization times (tpoly).
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Figure 2: A reinitiated φ6 RdRP elongation complex (φ6EC) and randomly-initiated φ6 RdRP replication show distinct electrophoretic profiles on agarose gels. (A) Schematic of stalling and reinitiation of φ6EC. In the presence of three NTPs (ATP, GTP, CTP), a φ6EC is stalled at the 50th nt from the 3′-end of the template at temperature T1. The sequence elongated prior to stalling is shown in blue. After UTP addition, the stalled φ6EC reinitiates and synthesizes the complementary strand at temperature T2. (B) Agarose gel of the elongation intermediates after reinitiation of the stalled φ6EC on 4 kb ssRNA template. Aliquots were taken at different times after reinitiation (telong). Letters S and P indicate 4 kb ssRNA and 4 kb dsRNA, respectively. (C) Schematic of a randomly-initiated φ6 RdRP replication. 4 kb ssRNA was incubated with φ6 RdRP, and all four NTPs were subsequently added simultaneously. (D) Agarose gel of aliquots of randomly-initiated φ6 RdRP replication taken at different polymerization times (tpoly).
Mentions: In replication, a φ6 RdRP elongation complex (φ6EC) can be stalled in vitro using a limited selection of NTPs and a template molecule in which the 3′-terminal region is devoid of one or more of the nucleotides (Figure 2A; for proof of stalling on short oligos, see Supplementary Data and Figure S3). Elongation can then be reinitiated by the addition of the missing NTP(s), yielding an entirely double-stranded product. To study the effect of stalling on the rate of φ6 RdRP replication, we selected a 4193 nt long replication template (4 kb ssRNA) in which the first occurrence of adenine was 50 nt from the 3′-end. The stalled φ6EC exhibited an electrophoretic mobility indistinguishable from that of free 4 kb ssRNA (Supplementary Figure S4). Following stalling and reinitiation by UTP addition, aliquots were collected at successive time points and analyzed on agarose gel (Figure 2B). After reinitiation, a fraction of the replication template retained the electrophoretic mobility of free 4 kb ssRNA (Supplementary Figure 2B, lanes 2–10), which corresponds to either free 4 kb ssRNA or to inactive stalled φ6EC. The electrophoretic mobility of successfully reinitiated φ6ECs decreased with time as φ6 RdRP progressed along the 4 kb ssRNA (Figure 2B, lanes 2–10). Notably, the band corresponding to the reinitiated φ6ECs stayed well-defined, suggesting that the stalled φ6ECs reinitiated in a synchronized manner. Termination of the replication reaction was detected by a stabilization of the electrophoretic mobility of the reinitiated φ6ECs (data not shown). This occurred between 30 min and 60 min after reinitiation, from which we deduce an overall kelong between 1.2 and 2.3 nt·s−1. (For the discussion of the measured rates please see Supplementary Data).Figure 2.

Bottom Line: Here, we report that RdRP experience a dramatic, long-lived decrease in its elongation rate when it is reinitiated following stalling.The rate decrease has an intriguingly weak temperature dependence, is independent of both the nucleotide concentration during stalling and the length of the RNA transcribed prior to stalling; however it is sensitive to RNA structure.This allows us to delineate the potential factors underlying this irreversible conversion of the elongation complex to a less active mode.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Applied Sciences, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.

ABSTRACT
RNA-dependent RNA polymerases (RdRP) form an important class of enzymes that is responsible for genome replication and transcription in RNA viruses and involved in the regulation of RNA interference in plants and fungi. The RdRP kinetics have been extensively studied, but pausing, an important regulatory mechanism for RNA polymerases that has also been implicated in RNA recombination, has not been considered. Here, we report that RdRP experience a dramatic, long-lived decrease in its elongation rate when it is reinitiated following stalling. The rate decrease has an intriguingly weak temperature dependence, is independent of both the nucleotide concentration during stalling and the length of the RNA transcribed prior to stalling; however it is sensitive to RNA structure. This allows us to delineate the potential factors underlying this irreversible conversion of the elongation complex to a less active mode.

Show MeSH
Related in: MedlinePlus