Abstract
Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.
Highlights
Programmed -1 frameshifting in SARS-related coronaviruses occurs at the slippery sequence U_UUA_AAC in the context of a 3′ stimulatory RNA sequence that was predicted to form a 3-stemmed pseudoknot structure [5], and in parallel was independently tested by our lab and others [6,7,8]
First release: 13 May 2021 www.sciencemag.org (Page numbers not final at time of first release) 1 mapping, in vivo selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), nuclear magnetic resonance (NMR) and cryo-electron microscopy [7, 14,15,16,17]
The cryo-electron microscopy (cryo-EM) 3D reconstruction of ribosome-nascent chain complexes (RNCs) affinity purified from the reactions supplemented with eRF1 (AAQ) revealed two distinct ribosomal complexes captured in the process of translating the slippery sequence
Summary
Programmed -1 frameshifting in SARS-related coronaviruses occurs at the slippery sequence U_UUA_AAC in the context of a 3′ stimulatory RNA sequence that was predicted to form a 3-stemmed pseudoknot structure [5], and in parallel was independently tested by our lab and others [6,7,8]. To provide a structural and mechanistic description of the events during ribosomal frameshifting, we investigated mammalian ribosomes captured in distinct functional states during translation of a region of SARS-CoV-2 genomic RNA where -1 programmed frameshifting occurs.
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