Puromycin reveals a distinct conformation of neuronal ribosomes

Abstract

Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and this event is normally followed by the dissociation of the puromycylated peptide. It was postulated that blocking the translocation of the ribosome with emetine could trap the puromycylated peptide on the ribosome, but evidence against this has recently been published (Hobson et al., 2020 https://doi.org/10.7554/eLife.60048; Enam et al., 2020 https://doi.org/10.7554/eLife.60303). In neurons, puromycylated nascent chains appear to remain in the ribosome even in the absence of emetine, yet direct evidence for this had been lacking. Using biochemistry and cryo-electron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state ribosomes, anisomycin, which normally blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains in neurons stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide new insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.

Puromycin reveals a distinct conformation of neuronal ribosomes

“…Cryo-EM map visualization was performed in UCSF Chimera ( 49 ) and Chimera X ( 52 ). The structures are available on the EMDB (Electron Microscopy Data Bank) ( https://www.ebi.ac.uk/emdb/ ; accession codes in Table 1 ) ( 56 – 59 ).…”