Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We use a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide evidence that the RNA-dependent RNA polymerase (RdRp) uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enable the direct observation of coronavirus polymerase deep and long-lived backtracking that is strongly stimulated by secondary structures in the template. The framework we present here elucidates one of the most important structure-dynamics-function relationships in human health today and will form the grounds for understanding the regulation of this complex.
|Number of pages||23|
|Early online date||17 Aug 2021|
|Publication status||Published - 31 Aug 2021|
Bibliographical noteFunding Information:
D.D. thanks OICE for hosting his lab. D.D. was supported by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg, the German Research Foundation ( DFG-DU-1872/3-1 ), the Netherlands Ministry of Education, Culture and Science (OCW) BaSyC (Building a Synthetic Cell) Gravitation grant ( 024.003.019 ), and the Netherlands Organisation for Scientific Research (NWO). R.N.K. was supported by NIAID NIH ( AI123498 ). B.C. acknowledge grants by the Fondation pour la Recherche Médicale (Aide aux équipes), the SCORE project H2020 SC1-PHE-Coronavirus-2020 ( 101003627 ), and the REACTing initiative (REsearch and ACTion targeting emerging infectious diseases). J.J.A. and C.E.C. were supported by NIAID NIH ( AI045818 ).
© 2021 The Author(s)
- high-throughput/ultra-stable magnetic tweezers
- nucleotide addition cycle
- polymerase mechanochemistry
- SARS-CoV-2 polymerase
- single-molecule biophysics