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| Title | Puromycin reveals a distinct conformation of neuronal ribosomes. |
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| Journal, issue, pages | Proc Natl Acad Sci U S A, Vol. 121, Issue 7, Page e2306993121, Year 2024 |
| Publish date | Feb 13, 2024 |
 Authors | Mina N Anadolu / Jingyu Sun / Jewel T-Y Li / Tyson E Graber / Joaquin Ortega / Wayne S Sossin /  |
| PubMed Abstract | Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It ...Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson , , e60048 (2020); and Enam , , e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron 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 neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide 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. |
 External links | Proc Natl Acad Sci U S A / PubMed:38315848 / PubMed Central |
| Methods | EM (single particle) |
| Resolution | 3.1 - 3.3 Å |
| Structure data |  EMDB-40665: Puromycin reveals a distinct conformation of neuronal ribosomes.  EMDB-40666: Class2 80S with PP tRNA  EMDB-40667: Class3 80S with broken density in P site  EMDB-40668: Class4 80S with only 60S |
| Source |
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Rattus (rat)