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Updated: Jul 4, 2025
Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
Published on: February 25, 2011
Puromycin reveals a distinct conformation of neuronal ribosomes
Mina N Anadolu1, Jingyu Sun2,3, Jewel T-Y Li1
1Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC H3A 2B4, Canada.
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 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 et al., Elife 9, e60048 (2020); and Enam et al., Elife 9, 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.
Insights
Puromycin-labeled nascent peptides remain stalled in neuronal ribosomes, even without emetine. This finding, visualized with cryo-EM, reveals mechanisms of neuronal ribosome stalling and offers a new assay for stalled peptide detection.
Area of Science:
- Molecular Biology
- Neuroscience
- Structural Biology
Background:
- Puromycin labeling of nascent peptide chains is a standard technique, typically followed by dissociation from the ribosome.
- Previous studies suggested emetine could prevent this dissociation, but recent evidence contradicts this.
- In neurons, puromycylated peptides appear to remain on ribosomes without emetine, though direct evidence was lacking.
Purpose of the Study:
- To provide direct evidence for puromycylated peptides remaining on neuronal ribosomes.
- To investigate the mechanism and prevalence of ribosome stalling in neurons.
- To develop a novel assay for identifying stalled neuronal ribosomes.
Main Methods:
- Biochemical assays to study puromycylation and ribosome stalling.
- Cryo-electron microscopy (cryo-EM) to visualize ribosome structure.
- Analysis of hippocampal neuronal cultures to quantify stalled polysomes.
Main Results:
- Puromycylated peptides were observed to remain within the ribosome exit channel of the large subunit in a subset of neuronal ribosomes.
- These stalled ribosomes were identified in a hybrid state.
- Anisomycin showed poor competition with puromycin in these stalled ribosomes, enabling a new assay; over 50% of nascent peptides in hippocampal neurons were found stalled.
Conclusions:
- Neuronal ribosomes can stall in a hybrid state, retaining puromycylated peptides in the exit channel.
- This study validates previous localization of stalled polysomes and offers insights into neuronal ribosome stalling mechanisms.
- Puromycylated peptides serve as a marker for identifying subcellular locations of hybrid-state stalled ribosomes in neurons.
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