Elongation-Competent Pauses Govern the Fidelity of a Viral RNA-Dependent RNA Polymerase

Dulin, David; Vilfan, Igor D.; Berghuis, Bojk A.; Hage, Susanne; Bamford, Dennis H.; Poranen, Minna M.; Depken, Martin; Dekker, Nynke H.

doi:10.1016/j.celrep.2015.01.031

Cited by 76 publications

(181 citation statements)

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“…The assay builds on similar arrangements developed to study processive enzymes, such as the T7 DNA polymerase (27,28), the helicase UrvD (29), λ exonuclease (30), and an RNA-dependent RNA polymerase (31). Here, we extend the assay to study processive exoribonuclease motion with high spatial (near-nucleotide) resolution.…”

mentioning

confidence: 99%

Direct observation of processive exoribonuclease motion using optical tweezers

Fazal

Koslover

Luisi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial RNases catalyze the turnover of RNA and are essential for gene expression and quality surveillance of transcripts. In Escherichia coli, the exoribonucleases RNase R and polynucleotide phosphorylase (PNPase) play critical roles in degrading RNA. Here, we developed an optical-trapping assay to monitor the translocation of individual enzymes along RNA-based substrates. Single-molecule records of motion reveal RNase R to be highly processive: one molecule can unwind over 500 bp of a structured substrate. However, enzyme progress is interrupted by pausing and stalling events that can slow degradation in a sequence-dependent fashion. We found that the distance traveled by PNPase through structured RNA is dependent on the A+U content of the substrate and that removal of its KH and S1 RNA-binding domains can reduce enzyme processivity without affecting the velocity. By a periodogram analysis of single-molecule records, we establish that PNPase takes discrete steps of six or seven nucleotides. These findings, in combination with previous structural and biochemical data, support an asymmetric inchworm mechanism for PNPase motion. The assay developed here for RNase R and PNPase is well suited to studies of other exonucleases and helicases.single-molecule biophysics | optical trap | exoribonuclease | processivity | step size B oth polynucleotide phosphorylase (PNPase) and RNase R are responsible for degrading a variety of transcripts in bacteria, such as mRNAs, defective rRNAs, and antisense regulatory RNAs (1-4). Although strains of Escherichia coli remain viable with a loss of either enzyme, the elimination of both results in an accumulation of rRNA fragments and cell death (3). To degrade RNA, both ribonucleases require an unstructured binding site of at least 7-10 nt at the 3′ end of the substrate (5-7), a sequence that often is supplied by posttranscriptional polyadenylation (8-10). After binding RNA, PNPase and RNase R processively digest the substrate in the 3′-to-5′ direction (11-13).PNPase consists of a trimer that degrades RNA at catalytic sites within a central channel (9,14). The enzyme is homologous to eukaryotic and archaeal exosomes and, like these hexameric proteins, possesses a core of six RNase PH domains arranged in a ringlike configuration (two per protomer) (6, 15). Each protomer also carries a KH and an S1 RNA-binding domain at its C terminus, both of which are connected to the body of the enzyme by flexible linkers (6, 16). These domains are thought to bind RNA upstream of the catalytic sites, and recent structural evidence suggests that they may guide the substrate into the central channel through a "hands gripping a rope" mechanism (16,17). By itself, PNPase may stall on structured RNA, particularly on G:C hairpins comprising more than six base pairs (18). PNPase often associates with the DEAD-box helicase RhlB to move through structured regions and generally does so in the context of the multienzyme degradosome (2, 19). However, there is some evidence that PNPase can digest structured tra...

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mentioning

confidence: 99%

Direct observation of processive exoribonuclease motion using optical tweezers

Fazal

Koslover

Luisi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…As mentioned above, the power-law distribution at longer dwell-times corresponds to back-tracking events which in the experimental setup for single molecule measurements lead to reannealing of the tethered dsRNA (Dulin et al, 2015b; Dulin et al, 2015a). This reversal of the direction of motion has also seen in bacterial and eukaryotic RNA polymerases (Shaevitz et al, 2003).…”

Section: Elongation At the Single-molecule Levelmentioning

confidence: 95%

“…al. (Dulin et al, 2015b) have studied elongation at the single molecule level using a clever setup utilizing magnetic tweezers (Figure 10). A length of dsRNA is tethered to a surface at one end to a magnetic bead on the other.…”

Section: Elongation At the Single-molecule Levelmentioning

confidence: 99%

“…The lower panel depicts a dwell time distribution (DTD) plot displaying four regions: rapid addition without pause (a Γ-distribution), two sets of short pauses (Pause 1 and Pause 2 described by exponential distributions) and a power-law distribution representing long pauses due to back-tracking. Modified from (Dulin et al, 2015b) with permission.…”

Section: Figurementioning

confidence: 99%

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Cystoviral RNA-directed RNA polymerases: Regulation of RNA synthesis on multiple time and length scales

Alphonse

Ghose

2017

Virus Research

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P2, an RNA-directed RNA polymerase (RdRP), is encoded on the largest of the three segments of the double-stranded RNA genome of cystoviruses. P2 performs the dual tasks of replication and transcription de novo on single-stranded RNA templates, and plays a critical role in the viral life-cycle. Work over the last few decades has yielded a wealth of biochemical and structural information on the functional regulation of P2, on its role in the spatiotemporal regulation of RNA synthesis and its variability across the Cystoviridae family. These range from atomic resolution snapshots of P2 trapped in functionally significant states, in complex with catalytic/structural metal ions, polynucleotide templates and substrate nucleoside triphosphates, to P2 in the context of viral capsids providing structural insight into the assembly of supramolecular complexes and regulatory interactions therein. They include in vitro biochemical studies using P2 purified to homogeneity and in vivo studies utilizing infectious core particles. Recent advances in experimental techniques have also allowed access to the temporal dimension and enabled the characterization of dynamics of P2 on the sub-nanosecond to millisecond timescale through measurements of nuclear spin relaxation in solution and single molecule studies of transcription from seconds to minutes. Below we summarize the most significant results that provide critical insight into the role of P2 in regulating RNA synthesis in cystoviruses.

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“…Based on the myriad RdRp fidelity mutants that have been reported to date, there appears to be a direct correlation between the rate of nucleotide addition (speed) and the fidelity of nucleotide addition [7,11,18,23,43]. This correlation also extends to recombination efficiency [23].…”

Section: Biochemical Properties Of the Rdrp Other Than Speed And Fidementioning

confidence: 99%

RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination

Kim

Ellis

Woodman

et al. 2019

Genes

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Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the KH virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination. Although the IF-PS-KH RdRp exhibits biochemical properties within twofold of wild type, the virus is impaired substantially for recombination in cells. We conclude that there are biochemical properties of the RdRp in addition to speed and fidelity that determine the mechanism and efficiency of recombination. The interwoven nature of speed, fidelity, the undefined property suggested here, and recombination makes it impossible to attribute a single property of the RdRp to fitness. However, the derivatives described here may permit elucidation of the importance of recombination on the fitness of the viral population in a background of constant polymerase speed and fidelity.broad-spectrum therapeutics [2-4] and a target for function/mechanism-based strategies for viral attenuation [5-10]. One function/mechanism of the RdRp that has been targeted most is that required for faithful incorporation of nucleotides [5][6][7][8][9][10]. Changing RdRp fidelity decreases or increases the genetic variation of the viral population, which, in turn, decreases fitness and virulence of the viral population [11][12][13][14][15][16].Known RdRp variants exhibiting a high-fidelity phenotype can also exhibit a reduced speed of nucleotide incorporation, at least at the biochemical level [17]. Replication speed is also a determinant of viral fitness and virulence [18]. So, is it the increased fidelity or decreased speed of nucleotide addition that gives rise to the attenuated phenotype? Further complicating the fidelity-versus-speed question are the recent observations that changes to fidelity also have consequence for the efficiency of recombination [19][20][21][22][23][24]. Increased RdRp fidelity decreases recombination efficiency and vice versa [19][20][21][22][23][24]. It will likely be impossible to attribute a single biochemical property of the RdRp to biological outcome.The most extensively studied PV fidelity mutants encode RdRps with amino acid substitutions at sites remote from the active site. Constructing equivalent substitutions conferring equivalent phenotypes in RdRps other than PV is difficult if not impossible. Our laboratory has therefore pursued active-site-based strategies to manipulate the fidelity, speed, and/or recombination efficiency of the RdRp [25]. We have shown that a lysine (Lys-359 in PV) present in ...

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Elongation-Competent Pauses Govern the Fidelity of a Viral RNA-Dependent RNA Polymerase

Cited by 76 publications

References 49 publications

Direct observation of processive exoribonuclease motion using optical tweezers

Direct observation of processive exoribonuclease motion using optical tweezers

Cystoviral RNA-directed RNA polymerases: Regulation of RNA synthesis on multiple time and length scales

RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination

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