Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and ...impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β-D-N
-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.
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 3'-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 3'-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 3'-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.
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
. ...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
. 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)
.
Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large ...multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double- and single-stranded DNA, consistent with their translocase and helicase activities, respectively. XPA releases the inhibitory kinase module of TFIIH, displaces a 'plug' element from the DNA-binding pore in XPD, and together with the NER factor XPG stimulates XPD activity. Our results explain how TFIIH is switched from a transcription to a repair factor, and provide the basis for a mechanistic analysis of the NER pathway.
Protein domains can fold into stable tertiary structures while they are synthesized on the ribosome. We used a high-performance, reconstituted in vitro translation system to investigate the folding ...of a small five-helix protein domain-the N-terminal domain of Escherichia coli N5-glutamine methyltransferase HemK-in real time. Our observations show that cotranslational folding of the protein, which folds autonomously and rapidly in solution, proceeds through a compact, non-native conformation that forms within the peptide tunnel of the ribosome. The compact state rearranges into a native-like structure immediately after the full domain sequence has emerged from the ribosome. Both folding transitions are rate-limited by translation, allowing for quasi-equilibrium sampling of the conformational space restricted by the ribosome. Cotranslational folding may be typical of small, intrinsically rapidly folding protein domains.
The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic ...subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs.
Transcription-coupled DNA repair removes bulky DNA lesions from the genome
and protects cells against ultraviolet (UV) irradiation
. Transcription-coupled DNA repair begins when RNA polymerase II ...(Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4
and UV-stimulated scaffold protein A (UVSSA)
. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published
data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, EC
, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4
spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4
lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.
Elongin is a heterotrimeric elongation factor for RNA polymerase (Pol) II transcription that is conserved among metazoa. Here, we report three cryo-EM structures of human Elongin bound to ...transcribing Pol II. The structures show that Elongin subunit ELOA binds the RPB2 side of Pol II and anchors the ELOB-ELOC subunit heterodimer. ELOA contains a 'latch' that binds between the end of the Pol II bridge helix and funnel helices, thereby inducing a conformational change near the polymerase active center. The latch is required for the elongation-stimulatory activity of Elongin, but not for Pol II binding, indicating that Elongin functions by allosterically regulating the conformational mobility of the polymerase active center. Elongin binding to Pol II is incompatible with association of the super elongation complex, PAF1 complex and RTF1, which also contain an elongation-stimulatory latch element.
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