Summary COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently being treated using Remdesivir, a nucleoside analog that inhibits the RNA-dependent-RNA polymerase (RdRp). However, the enzymatic mechanism and efficiency of Remdesivir have not been determined, and reliable screens for new inhibitors are urgently needed. Here we present our work to optimize expression in E. coli , followed by purification and kinetic analysis of an untagged NSP12/7/8 RdRp complex. Pre-steady-state kinetic analysis shows that our reconstituted RdRp catalyzes fast ( k cat = 240–680 s −1 ) and processive ( k off = 0.013 s −1 ) RNA polymerization. The specificity constant ( k cat /K m ) for Remdesivir triphosphate (RTP) incorporation (1.29 μM −1 s −1 ) is higher than that for the competing ATP (0.74 μM −1 s −1 ). This work provides the first robust analysis of RNA polymerization and RTP incorporation by the SARS-CoV-2 RdRp and sets the standard for development of informative enzyme assays to screen for new inhibitors. Graphical Abstract Highlights • Co-expression of NSP12/7/8 with chaperones in E. coli gives soluble SARS CoV2 RdRp • Tag-free RdRp complex catalyzes fast and processive RNA polymerization • Polymerization rates are sufficient to replicate the 30 kb genome in 2 min • Remdesivir is incorporated with a specificity constant twice that observed for ATP Biological Sciences; Biochemistry; Molecular Biology; Molecular Biology Experimental Approach
【저자키워드】 Biochemistry, Molecular biology, Biological sciences, Molecular Biology Experimental Approach, 【초록키워드】 COVID-19, SARS-CoV-2, coronavirus, Genome, inhibitors, RNA, specificity, Biology, RdRP, ATP, inhibitor, expression, mechanism, Analysis, Efficiency, Biological, acute respiratory syndrome, enzyme, complex, purification, polymerization, polymerase, chaperone, kinetic analysis, robust, caused, inhibit, replicate, treated, provide, competing, the SARS-CoV-2, 【제목키워드】 COVID-19, RNA, ATP, substrate, Better,