New preprint on SARS-CoV-2 polymerase mechanochemistry!

Congrats to all the authors of the Dulin lab (in particular Subhas and Mona) and to all the collaborators for their great work! Below is the Twitter thread describing the work. Link of the preprint here

Now the science: the coronavirus core polymerase is made of the RNA-dependent RNA polymerase #RdRp nsp12 and the co-factors nsp7 + two nsp8’s. It synthesizes all the viral RNA in infected cells and is absolutely key to virus survival and therefore a great drug target.

However, we don’t know much about the stability (must synthesize 30 kb long ssRNA!) and the kinetics of the complex. Nothing about elongation on a 1 kb long ssRNA template or with the presence of secondary structures on the template. We did it. This is what we found:

1. The complex is stable once assembled and can elongate a 1 kb long template without free viral protein in solution. The observed dynamics is therefore intrinsic to the polymerase activity (no viral protein exchange).

2. We observed the response of the #SARSCoV2 polymerase at various concentration of NTPs and applied tension to investigate the nucleotide addition cycle. Conclusions:

2.1 Nucleotide addition occurs through 3 separate pathways: nucleotide addition burst (NAB), and the slow and very slow nucleotide addition (SNA and VSNA) pathways. SNA and VSNA appear as short duration pauses (1-5 seconds);

2.2 Translocation is thermally activated and occurs at the beginning of the nucleotide addition cycle. The switch between the 3 catalytic pathways happens in the pre-translocated state. Important: NTP and nucleotide analogue binding do not trigger the switch;

2.3 Following NTP binding, the nucleotide addition cycle is followed by a succession of two (nearly) irreversible steps: chemistry and a large conformational change, likely a polymerase “reset” to enable translocation and the next cycle. True for NAB, SNA and VSNA.

3. Catalytically inactive long-lived pauses are strongly stimulated by template secondary structures. Improving magnetic tweezers stability, we show these pauses are consistent with polymerase #backtrack. The #SARSCoV2 polymerase backtracks when facing secondary structures.

3.1 Our results supports a recent structure of a pre-assembled backtracking #SARSCoV2 polymerase complex https://biorxiv.org/content/10.1101/2021.03.13.435256v1

3.2 #SARSCoV2 Polymerase backtrack is likely linked to viral genome recombination and transcription. See recent work from @CramerLabhttps://biorxiv.org/content/10.1101/2021.03.23.436644v1

3.2 This opens plenty of exciting questions: what is polymerase backtracking role in coronavirus replication/transcription? Secondary structures regulatory role? How does helicase nsp13 assist #SARSCoV2 polymerase to resolve them? And other co-factors, e.g. nsp9?