Dynamics of transcription and error incorporation in a viral RNA-dependent RNA polymerase
David Dulin (Delft University of Technology)
RNA-dependent RNA polymerases (RdRPs) are essential enzymes that govern transcription and replication in RNA viruses. While RNA elongation forms an important therapeutic target against viral infection, little is known about elongation dynamics at the single-molecule level. Here, we study the well-established RdRP model system of P2 from the double-stranded RNA bacteriophage Φ6 using high-throughput single-molecule force-spectroscopy combined with theoretical modeling. We show that P2 elongation dynamics is irregular, with rapid transcription repeatedly interrupted by pauses whose durations vary from seconds to thousands of seconds. Exploiting the discriminatory power offered by our large datasets, together with specifically-adapted analysis, we introduce a stochastic dynamical model of P2 transcriptional elongation. Our results imply that the majority of pauses result from nucleotide misincorporation, providing a direct link between RdRP dynamics and error rates—rates that offer potential drug targets, as they must be finely tuned to confer both genome stability between generations and adaptability to bypass host defense systems.