title: Reconstitution of a minimal ribosome-associated ubiquitination pathway with purified factors.
abstract: Ribosomes stalled on aberrant mRNAs engage quality control mechanisms that degrade the partially translated nascent polypeptide. Ubiquitination of the nascent protein is mediated by the E3 ligase Listerin via a mechanism involving ribosome subunit dissociation. Here, we reconstitute ribosome-associated ubiquitination with purified factors to define the minimal components and essential steps in this process. We find that the primary role of the ribosome splitting factors Hbs1, Pelota, and ABCE1 is to permit Listerin access to the nascent chain. Listerin alone can discriminate 60S- from 80S-nascent chain complexes to selectively ubiquitinate the former. Splitting factors can be bypassed by artificially removing the 40S subunit, suggesting that mere steric hindrance impedes Listerin recruitment. This was illustrated by a cryo-EM reconstruction of the 60S-Listerin complex that identifies a binding interface that clashes with the 40S ribosomal subunit. These results reveal the mechanistic logic of the core steps in a ribosome-associated quality control pathway.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK.
authors.forename: Sichen
authors.initials: S
authors.lastname: Shao
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK. Electronic address: rhegde@mrc-lmb.cam.ac.uk.
authors.forename: Ramanujan S
authors.initials: RS
authors.lastname: Hegde
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D001665:Binding Sites; D020285:Cryoelectron Microscopy; D057809:HEK293 Cells; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D008958:Models, Molecular; D010445:Peptide Elongation Factors / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D054657:Ribosome Subunits / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D018528:ATP-Binding Cassette Transporters; D008840:Microfilament Proteins; D010445:Peptide Elongation Factors; D012335:RNA, Ribosomal; D044767:Ubiquitin-Protein Ligases