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- W2068014766 abstract "RNA-dependent RNA Polymerases (RdRPs) are key players in the transcription and replication of RNA viruses. We study P2 of the bacteriophage Φ6 as a model system for structurally similar polymerases of e.g. positive ssRNA viruses such as Hepatitis C. During the different phases of the viral life cycle, P2 RdRP alternately transcribes or replicates viral RNA. Transcription produces infectious positive-strand ssRNA whilst replication is the last step of the virus maturation in which the dsRNA genome is restored. Benefiting from the parallelism afforded by magnetic tweezers, we simultaneously measure the transcription activity on tens of tethers while maintaining a resolution of 6 bases. In this way, we report on polymerase dynamics based on the analysis of an unusually large dataset (800 traces) taken under differing conditions of force and nucleotides concentration. This enables us to characterize P2 transcription elongation dynamics with unprecedented precision. Fits of this data to a kinetic model for polymerase activity via Maximum Likelihood estimation (MLE) reveal that the translocation step is insensitive to force and points to the existence of several types of off-pathway pause states in which the polymerase may be trapped. The associated pauses from short-lived exponentially-distributed pauses to long-lived pauses that follow a power law distribution over three decades and likely result from backtracking. By comparing our single-molecule data to the results of previous structural and biochemical studies on related RdRPs, we propose that the exponentially-distributed pauses are connected to the nucleotide selection process. Backtracking has not been previously observed for the P2 RdRP, and our experiments show how it can be problematic for an RdRP, e.g. preventing it from completing transcription or replication." @default.
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- W2068014766 date "2013-01-01" @default.
- W2068014766 modified "2023-10-17" @default.
- W2068014766 title "Backtracking and Other Off-Path Pauses Control the Dynamic of Viral RNA Dependent RNA Polymerases" @default.
- W2068014766 doi "https://doi.org/10.1016/j.bpj.2012.11.2022" @default.
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