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Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local me...

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Autores principales: Hobson, Benjamin D, Kong, Linghao, Hartwick, Erik W, Gonzalez, Ruben L, Sims, Peter A
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490010/
https://www.ncbi.nlm.nih.gov/pubmed/32844746
http://dx.doi.org/10.7554/eLife.60048
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author Hobson, Benjamin D
Kong, Linghao
Hartwick, Erik W
Gonzalez, Ruben L
Sims, Peter A
author_facet Hobson, Benjamin D
Kong, Linghao
Hartwick, Erik W
Gonzalez, Ruben L
Sims, Peter A
author_sort Hobson, Benjamin D
collection PubMed
description Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.
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spelling pubmed-74900102020-09-16 Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes Hobson, Benjamin D Kong, Linghao Hartwick, Erik W Gonzalez, Ruben L Sims, Peter A eLife Biochemistry and Chemical Biology Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation. eLife Sciences Publications, Ltd 2020-08-26 /pmc/articles/PMC7490010/ /pubmed/32844746 http://dx.doi.org/10.7554/eLife.60048 Text en © 2020, Hobson et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biochemistry and Chemical Biology
Hobson, Benjamin D
Kong, Linghao
Hartwick, Erik W
Gonzalez, Ruben L
Sims, Peter A
Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes
title Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes
title_full Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes
title_fullStr Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes
title_full_unstemmed Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes
title_short Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes
title_sort elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes
topic Biochemistry and Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490010/
https://www.ncbi.nlm.nih.gov/pubmed/32844746
http://dx.doi.org/10.7554/eLife.60048
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AT gonzalezrubenl elongationinhibitorsdonotpreventthereleaseofpuromycylatednascentpolypeptidechainsfromribosomes
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