The new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes 1-3. Here we present a cryo-electron microscopy structure of the SARS-CoV-2 RdRp in an active form that mimics the replicating enzyme. The structure comprises the viral proteins non-structural protein 12 (nsp12), nsp8 and nsp7, and more than two turns of RNA template-product duplex. The active-site cleft of nsp12 binds to the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the second turn of RNA. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged 'sliding poles'. These sliding poles can account for the known processivity of RdRp that is required for replicating the long genome of coronaviruses 3. Our results enable a detailed analysis of the inhibitory mechanisms that underlie the antiviral activity of substances such as remdesivir, a drug for the treatment of coronavirus disease 2019 (COVID-19) 4. Coronaviruses are positive-strand RNA viruses that pose a major health risk 1 : SARS-CoV-2 has caused a pandemic of the disease known as COVID-19 5,6. Coronaviruses use an RdRp complex for the replication of their genome and for the transcription of their genes 2,3. This RdRp complex is the target of nucleoside analogue inhibitors-in particular, remdesivir 7,8. Remdesivir inhibits the RdRp of multiple coronaviruses 9,10 , and shows antiviral activity in cell culture and animal models 11. Remdesivir is currently being tested in the clinic in many countries 12 and has recently been approved for emergency treatment of patients with COVID-19 in the United States 4. The RdRp of SARS-CoV-2 is composed of a catalytic subunit known as nsp12 13 as well as two accessory subunits, nsp8 and nsp7 3,14. The structure of this RdRp has recently been reported 15 ; it is highly similar to the RdRp of SARS-CoV 16 , a zoonotic coronavirus that spread into the human population in 2002 1. The nsp12 subunit contains an N-terminal nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain, an interface domain and a C-terminal RdRp domain 15,16. The RdRp domain resembles a right hand, comprising the fingers, palm and thumb subdomains 15,16 that are found in all single-subunit polymerases. Subunits nsp7 and nsp8 bind to the thumb, and an additional copy of nsp8 binds to the fingers domain 15,16. Structural information is also available for nsp8-nsp7 complexes 17,18. To obtain the structure of the SARS-CoV-2 RdRp in its active form, we prepared recombinant nsp12, nsp8 and nsp7 (Fig. 1a, Methods). When added to a minimal RNA hairpin substrate (Fig. 1b), the purified proteins gave rise to RNA-dependent RNA extension activity, which depended on nsp8 and nsp7 (Fig. 1c). We assembled and purified a stable RdRp-RNA complex with the use of a self-annealing RNA, and collected single-particle cryo-electron microscopy (cryo-EM) data (Ex...

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

After purification of EC* by size exclusion chromatography and mild crosslinking with 85 glutaraldehyde, we determined its cryo-EM structure at a nominal resolution of 3.1 Å (Fig. 2, 86 Supplementary Video 1, Extended Data Fig. 2j). 2D classification revealed densities on the 87 Pol II surface (Extended Data Fig. 3, 4; Extended Data Table 2) and resulted in a 3D 88 reconstruction from 374,964 particles. The core of Pol II extended to ~2.6 Å resolution. 89 Vos et al.,: Structure of activated transcription complex Pol II-DSIF-PAF-SPT6Elongation factors were resolved at lower resolutions (~12 Å for the most flexible domains), 90 and their corresponding densities were improved by focused classification and refinement 91 (Extended Data Figs. 3-5, Methods). This led to a total of eight cryo-EM density maps that 92 enabled us to fit available structures and homology models (Extended Data Fig. 3; 93 Supplementary Table 1). Modeling was aided by lysine crosslinking data (Extended Data 94 Fig. 6, Supplementary Tables 2-4). 225 unique crosslinks were detected in structured regions, 95 of which 210 fell into the permitted 30 Å range. The remaining 15 crosslinks formed between 96 mobile elements of the structure (Extended Data Fig. 6; Supplementary Table 2). 97 To complete the EC* structure, we determined the crystal structure of the isolated 98 human SPT6 tandem SH2 (tSH2) domain at 1.8 Å resolution, and unambiguously docked this 99 new structure into the corresponding density of EC* (Fig. 3, Methods, Extended Data Fig. 5f, 100 6f, 7, Extended Data Table 3). The resulting structure of EC* shows good stereochemistry 101 and lacks only mobile regions, including the terminal regions of PAF1 and LEO1, most of 102 CDC73, the acidic N-terminal region of SPT6, and the C-terminal extensions of SPT5, SPT6, 103 and CTR9 ( Supplementary Table 1). 104 105 PAF and SPT6 structure and contacts 106 DSIF, PAF, and SPT6 are modular proteins that coat the outer surface of Pol II (Fig. 2). DSIF 107 domains are arrayed around the Pol II cleft and RNA exit tunnel 4 . PAF extends along the RPB2 108 side and docks on the Pol II funnel. PAF is anchored to the external domains of RPB2 via its 109 PAF1-LEO1 dimerization module (Fig. 2b, c). The central PAF subunit CTR9 contains 19 110 tetratricopeptide repeats (TPRs; residues 41-750) that each form two antiparallel ɑ-helices ( Fig. 111 3a, Supplementary Table 6, Extended Data Fig. 5b). The CTR9 TPRs form a right-handed 112 superhelix that extends from the Pol II subunit RPB11 along RPB8 via the polymerase funnel 113 to the foot (Fig. 3a). The TPRs are followed by a pair of helices that create a 'vertex' and 114 connect to a prominent 'trestle' helix in CTR9 (CTR9 residues 807-892) (Extended Data Fig. 115 5c). The trestle extends ~100 Å from the Pol II foot to subunit RPB5 where downstream DNA 116 enters the Pol II cleft. The vertex and TPRs 13, 14, and 18 buttress the PAF subunit WDR61, 117 which forms a seven-bladed β-propeller 28 and faces away from Pol II (Fig. 3a, Extended Data 118 Fi...

Metazoan gene regulation often involves the pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy structure of a paused transcription elongation complex containing Sus scrofa Pol II and Homo sapiens DSIF and NELF at 3.2 Å resolution. The structure reveals a tilted DNA-RNA hybrid that impairs binding of the nucleoside triphosphate substrate. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. NELF prevents binding of the anti-pausing transcription elongation factor IIS (TFIIS). Additionally, NELF possesses two flexible 'tentacles' that can contact DSIF and exiting RNA. These results define the paused state of Pol II and provide the molecular basis for understanding the function of NELF during promoter-proximal gene regulation.

Chromatin remodelling factors change nucleosome positioning and facilitate DNA transcription, replication, and repair1. The conserved remodelling factor Chd12 can shift nucleosomes and induce a regular nucleosome spacing3–5. Chd1 is required for RNA polymerase II passage through nucleosomes6 and for cellular pluripotency7. Chd1 contains the DNA-binding domains SANT and SLIDE, a bilobal motor domain that hydrolyses adenosine triphosphate (ATP), and a regulatory double chromodomain. Here we report the cryo-electron microscopy (cryo-EM) structure of Chd1 from the yeast S. cerevisiae bound to a nucleosome at a resolution of 4.8 Å. Chd1 detaches two turns of DNA from the histone octamer and binds between the two DNA gyres in a state poised for catalysis. The SANT and SLIDE domains contact detached DNA around superhelical location (SHL) -7 of the first DNA gyre. The ATPase motor binds the second DNA gyre at SHL +2 and is anchored to the N-terminal tail of histone H4 as in a recent nucleosome-Snf2 ATPase structure8. Comparison with published results9 reveals that the double chromodomain swings towards nucleosomal DNA at SHL +1, resulting in ATPase closure. The ATPase can then promote translocation of DNA towards the nucleosome dyad, thereby loosening the first DNA gyre and remodelling the nucleosome. Translocation may involve ratcheting of the two lobes of the ATPase, which is trapped in a pre- or post-translocated state in the absence8 or presence, respectively, of transition state-mimicking compounds.