Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site.
Transcription in RNA viruses is highly dynamic, with a variety of pauses interrupting nucleotide addition by RNA-dependent RNA polymerase (RdRp). For example, rare but lengthy pauses (>20 s) have been linked to backtracking for viral single-subunit RdRps. However, while such backtracking has been well characterized for multi-subunit RNA polymerases (RNAPs) from bacteria and yeast, little is known about the details of viral RdRp backtracking and its biological roles. Using high-throughput magnetic tweezers, we quantify the backtracking by RdRp from the double-stranded (ds) RNA bacteriophage Φ6, a model system for RdRps. 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.
Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
David
D
Dulin
Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Igor D
ID
Vilfan
Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Bojk A
BA
Berghuis
Department of Biosciences, University of Helsinki, Viikki Biocenter 1, P.O. Box 56 (Viikinkaari 9), 00014 Helsinki, Finland.
Minna M
MM
Poranen
Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Martin
M
Depken
Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Nynke H
NH
Dekker
D017126:Bacteriophage phi 6 / Q000201:enzymology*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D013698:Templates, Genetic; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D012324:RNA-Dependent RNA Polymerase