TY - JOUR
T1 - The nucleotide addition cycle of the SARS-CoV-2 polymerase
AU - Bera, Subhas Chandra
AU - Seifert, Mona
AU - Kirchdoerfer, Robert N.
AU - van Nies, Pauline
AU - Wubulikasimu, Yibulayin
AU - Quack, Salina
AU - Papini, Flávia S.
AU - Depken, Martin
AU - Dulin, David
AU - More Authors, null
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - backtracking
KW - high-throughput/ultra-stable magnetic tweezers
KW - nucleotide addition cycle
KW - polymerase mechanochemistry
KW - SARS-CoV-2 polymerase
KW - single-molecule biophysics
UR - http://www.scopus.com/inward/record.url?scp=85113701019&partnerID=8YFLogxK
U2 - 10.1016/j.celrep.2021.109650
DO - 10.1016/j.celrep.2021.109650
M3 - Article
AN - SCOPUS:85113701019
SN - 2211-1247
VL - 36
JO - Cell Reports
JF - Cell Reports
IS - 9
M1 - 109650
ER -