The Nucleotide Addition Cycle of the SARS-CoV-2 Polymerase

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 have used 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 the first evidence that an RdRp uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enables the direct observation of coronavirus polymerase deep and long-lived backtrack that are strongly stimulated by secondary structure in the template. The framework presented 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. Funding: DD was supported by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg, the German Research Foundation grant DFG-DU-1872/3-1 and BaSyC – Building a Synthetic Cell” Gravitation grant (024.003.019) of the Netherlands Ministry of Education, Culture and Science (OCW) and the Netherlands Organisation for Scientific Research (NWO). DD thanks OICE for providing office, lab space, and access to their molecular biology lab. RNK was supported by grant AI123498 from NIAID, NIH. BC acknowledge grants by the Fondation pour la Recherche Medicale (Aide aux equipes), the SCORE project H2020 SC1-PHE-Coronavirus-2020 (grant#101003627), and the REACTing initiative (REsearch and ACTion targeting emerging infectious diseases). JJA and CEC were supported by grant AI045818 from NIAID, NIH. Declaration of Interest: The authors declare no competing interest.