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

• Published in: Cell reports (Cambridge) 2021-08, Vol.36 (9), p.109650-109650, Article 109650
• Main Authors: Bera, Subhas Chandra, 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
• Format: Article
• Language: English
• Subjects: backtracking; Coronavirus RNA-Dependent RNA Polymerase - chemistry; Coronavirus RNA-Dependent RNA Polymerase - physiology; COVID-19 - virology; High-Throughput Screening Assays; high-throughput/ultra-stable magnetic tweezers; Humans; Models, Molecular; Molecular Conformation; nucleotide addition cycle; Nucleotides - chemistry; Nucleotides - metabolism; polymerase mechanochemistry; RNA, Viral - biosynthesis; RNA, Viral - chemistry; SARS-CoV-2 - physiology; SARS-CoV-2 polymerase; Single Molecule Imaging; single-molecule biophysics; Viral Nonstructural Proteins - chemistry; Viral Nonstructural Proteins - physiology
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2021.109650

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. [Display omitted] •The SARS-CoV-2 polymerase is a stable and processive RNA polymerase•The SARS-CoV-2 polymerase uses one fast and two slow nucleotide addition pathways•The nucleotide addition cycle starts with a thermally activated translocation•RNA secondary structures induce deep SARS-CoV-2 polymerase backtrack Bera et al. reveal the complete nucleotide addition cycle of the SARS-CoV-2 polymerase and show that the SARS-CoV-2 polymerase is a processive RNA polymerase that backtracks when elongating through RNA template containing secondary structures.