The nucleotide addition cycle of the SARS-CoV-2 polymerase

Chandra Bera, Subhas; Seifert, Mona; Kirchdoerfer, Robert N.; van Nies, Pauline; Wubulikasimu, Yibulayin; Quack, Salina; Papini, Flávia S.; Arnold, Jamie J.; Canard, Bruno; Cameron, Craig E.; Depken, Martin; Dulin, David

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 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 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.