Journal: Nucleic Acids Research

Article Title: A post-assembly conformational change makes the SARS-CoV-2 polymerase elongation-competent

doi: 10.1093/nar/gkaf450

Figure Lengend Snippet: Slow activation by reconstituted SARS-CoV-2 core RTCs. ( A ) Schematic of the magnetic tweezers assay to monitor activation and elongation by the SARS-CoV-2 core RTC (“Materials and methods” section). ( B ) Schematic representation different methods of obtaining the core RTC used throughout this work. (I) nsp7, nsp8, and nsp12-polymerase were separately and recombinantly expressed in Escherichia coli , (II) pull-down of a core RTC complex after expressing a bacmid containing nsp5-Mpro (main protease), nsp8, nsp7, and nsp12-polymerase in Sf9 cells, (III) nsp7, nsp8, and nsp12-polymerase were separately and recombinantly expressed in E. coli , and nsp12-polymerase was recombinantly expressed in Sf9 (“Materials and methods” section). Color coding for the different core RTCs is kept in subsequent figures to represent to related data. ( C ) Example time traces for the reconstituted SARS-CoV-2 core RTC using 0.16 μM nsp12-polymerase [see (I) in panel B] and 1.8 μM of nsp7 and nsp8. The orange (left-most) and purple (right-most) dashed lines indicate the end of the activation and elongation phases. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${\mathrm{\Delta }}{{t}_{\mathrm{A}}}$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${\mathrm{\Delta }}{{t}_{\mathrm{E}}}$\end{document} are their respective durations. ( D ) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${\mathrm{\Delta }}{{t}_{\mathrm{A}}}$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${\mathrm{\Delta }}{{t}_{\mathrm{E}}}$\end{document} for all recorded traces in experiments of which representative subsets are shown in panels (C), (E), and (F). Horizontal markers indicate the group medians. ( E ) Example time traces using 0.8 μM of the pulled-down SARS-CoV-2 core RTC [see (II) in panel B]. ( F ) Example time traces for the reconstituted SARS-CoV-2 core RTC using 0.25 μM nsp12-polymerase expressed in Sf9 [see (III) in panel B] and 1.8 μM of nsp7 and nsp8.

Article Snippet: The coding sequence for nsp13 from the SARS CoV-2 Washington isolate (Genbank MN985325 ) was synthesized as an E. coli codon-optimized fragment (GenScript, Piscataway NJ) and cloned into the Bsa I site of the pSUMO plasmid (LifeSensors, Malvern, PA) to produce an N -terminal six histidine-tagged SUMO-nsp13 fusion cassette (6XHis-SUMO-nsp13).

Techniques: Activation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: A post-assembly conformational change makes the SARS-CoV-2 polymerase elongation-competent

doi: 10.1093/nar/gkaf450

Figure Lengend Snippet: Presence of the template RNA reduces activation times. Example time traces are shown for a reconstituted core RTC. ( A ) Nsps were added together 4.5 hours (nsp12-polymerase expressed in E. coli ) or 6.5 h (nsp12-polymerase expressed in Sf9 ) prior to diluting them to 0.2 μM nsp12, 1.8 μM nsp7, and nsp8 and performing primer-extensions. In panels (A) and (B), percentages of rapidly activating polymerases represent the fraction of traces in which activity started during phase 1 (flushing the reagents into the flow chamber). ( B ) (1) The proteins were added to the RNA tethering the magnetic beads in the flow chamber. (2) Ten minutes later 500 μl of reaction buffer was used to rinse the flow chamber from free-floating proteins. (3) Finally, ribonucleotides were added and (4) activation typically started during step (3). ( C ) (1) After tethering the magnetic beads to the RNA, 0.2 μM of nsp12-polymerase, and 500 μM of NTP were added. Ten minutes later 500 μl of reaction buffer was used to remove free-floating proteins. (3) The nucleotides were reintroduced together with 1.8 μM of nsp7 and nsp8. (4) RTC elongation activity.

Article Snippet: The coding sequence for nsp13 from the SARS CoV-2 Washington isolate (Genbank MN985325 ) was synthesized as an E. coli codon-optimized fragment (GenScript, Piscataway NJ) and cloned into the Bsa I site of the pSUMO plasmid (LifeSensors, Malvern, PA) to produce an N -terminal six histidine-tagged SUMO-nsp13 fusion cassette (6XHis-SUMO-nsp13).

Techniques: Activation Assay, Activity Assay, Magnetic Beads

Journal: Nucleic Acids Research

Article Title: A post-assembly conformational change makes the SARS-CoV-2 polymerase elongation-competent

doi: 10.1093/nar/gkaf450

Figure Lengend Snippet: The activation phase and the mode of core RTC proteins expression do not influence the kinetics of the elongating core RTC. ( A ) Example time traces during elongation phase of either reconstituted core RTC with 0.16 μM nsp12-polymerase expressed in E. coli , and 1.8 μM nsp7 and nsp8 (turquoise), reconstituted core RTC with 0.2 μM nsp12-polymerase expressed in Sf9 and 1.8 μM nsp7 and nsp8 (purple), or 2 nM of pulled-down core RTC (yellow). ( B ) Mean elongation times ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${\mathrm{\Delta }}{{t}_{\mathrm{E}}}$\end{document} ) and product lengths ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${\mathrm{\Delta }}{{L}_{\mathrm{E}}})$\end{document} , and ( C ) dwell time distributions for the entire set of activity traces recorded. The solid line in panel (C) represents the distribution we previously reported in . ( D ) Example time traces during elongation phase of a reconstituted core RTC (nsp12-polymerase expressed in E. coli ) at 1.8 μM (black) and 0.2 μM (gray) of nsp7. ( E ) Mean elongation times and product lengths, and ( F ) dwell time distributions across nsp7 concentrations. ( G ) Example time traces during elongation of reconstituted core RTC (nsp12-polymerase expressed in E. coli ) at 1.8 μM (black) and 0.6 μM (gray) of nsp8. ( H ) Mean elongation times and product lengths, and ( I ) dwell time distribution across nsp8 concentrations. All error bars represent 95% confidence intervals determined as described in “Materials and methods” section.

Article Snippet: The coding sequence for nsp13 from the SARS CoV-2 Washington isolate (Genbank MN985325 ) was synthesized as an E. coli codon-optimized fragment (GenScript, Piscataway NJ) and cloned into the Bsa I site of the pSUMO plasmid (LifeSensors, Malvern, PA) to produce an N -terminal six histidine-tagged SUMO-nsp13 fusion cassette (6XHis-SUMO-nsp13).

Techniques: Activation Assay, Expressing, Activity Assay

Journal: Nucleic Acids Research

Article Title: A post-assembly conformational change makes the SARS-CoV-2 polymerase elongation-competent

doi: 10.1093/nar/gkaf450

Figure Lengend Snippet: Assembly and RNA binding of the core RTC cannot fully account for the observed activation times. When not being varied, experiments were performed using 0.16 μM of nsp12-polymerase, 1.8 μM nsp7, 1.8 μM nsp8, and 25 pN. Effective activation times across ( A ) nsp7 concentration, ( B ) nsp8 concentration, and ( C ) nsp12-polymerase concentration (expressed in E. coli ). ( D ) Activation times versus nsp12-polymerase concentration, while maintaining a constant stoichiometry of [nsp12-polymerase]:[nsp7]:[nsp8] as 1:9:9 in solution. Core RTC reconstituted using nsp12-polymerase expressed in E. coli (turquoise triangles) or Sf9 (purple squares). ( E ) Activation times versus concentration of pulled-down core RTC. ( F ) Activation times versus force (reconstituted core RTC, all proteins expressed in E. coli ). Error bars represent 95% confidence intervals estimated through bootstrapping (“Materials and methods” section). Effective activation times shown incorporate the fraction of events lasting longer than the recording (“Materials and methods” section). Solid curves are the best fit of our mechanochemical model (“Materials and methods” section, Fig. and ). Dashed horizontal lines are shown to guide-the-eye.

Article Snippet: The coding sequence for nsp13 from the SARS CoV-2 Washington isolate (Genbank MN985325 ) was synthesized as an E. coli codon-optimized fragment (GenScript, Piscataway NJ) and cloned into the Bsa I site of the pSUMO plasmid (LifeSensors, Malvern, PA) to produce an N -terminal six histidine-tagged SUMO-nsp13 fusion cassette (6XHis-SUMO-nsp13).

Techniques: RNA Binding Assay, Activation Assay, Concentration Assay