Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein crosslinking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3'-segment of the product-RNA generated by backtracking extrudes through the RdRp NTP-entry tunnel, that a mismatched nucleotide at the product-RNA 3'-end frays and enters the NTP-entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.

Download data

• Downloaded 562 times
• Download rankings, all-time:
  • Site-wide: 50,184
  • In biochemistry: 1,304
• Year to date:
  • Site-wide: 5,182
• Since beginning of last month:
  • Site-wide: 18,052

Altmetric data

Downloads over time

Distribution of downloads per paper, site-wide