Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site

Nucleic Acids Res. 2015 Dec 2;43(21):10421-9. doi: 10.1093/nar/gkv1098. Epub 2015 Oct 22.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophage phi 6 / enzymology*
  • RNA-Dependent RNA Polymerase / metabolism*
  • Templates, Genetic
  • Transcription Initiation Site*
  • Transcription, Genetic

Substances

  • RNA-Dependent RNA Polymerase