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

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Benjamin D Hobson ◽  
Linghao Kong ◽  
Erik W Hartwick ◽  
Ruben L Gonzalez ◽  
Peter A Sims

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.

Author(s):  
Benjamin D. Hobson ◽  
Linghao Kong ◽  
Erik W. Hartwick ◽  
Ruben L. Gonzalez ◽  
Peter A. Sims

ABSTRACTPuromycin 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, 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.


Blood ◽  
2014 ◽  
Vol 123 (6) ◽  
pp. 905-913 ◽  
Author(s):  
Erika K. Keeton ◽  
Kristen McEachern ◽  
Keith S. Dillman ◽  
Sangeetha Palakurthi ◽  
Yichen Cao ◽  
...  

Key Points AZD1208 is a selective pan-Pim kinase inhibitor with efficacy in AML cells, xenografts, and Flt3-internal tandem duplication or Flt3 wild-type patient samples. AML cell growth inhibition is associated with suppression of p70S6K, 4EBP1 phosphorylation, and messenger RNA translation.


Development ◽  
1965 ◽  
Vol 13 (3) ◽  
pp. 341-356
Author(s):  
F. S. Billett ◽  
Rosalba Collini ◽  
Louie Hamilton

In many bacterial systems chloramphenicol has been shown to inhibit protein synthesis (Hahn & Wisseman, 1951; Gale & Folkes, 1953). The precise mechanism of this inhibition is not clear, although the evidence suggests that the interaction of the soluble RNA-amino acid complex with the ribosomes is prevented because the attachment of the messenger RNA to the ribosomes is itself impaired (Lacks & Gros, 1959; Nathans & Lipman, 1961; Jardetsky & Julian, 1964; Julian & Jardetsky, 1964). In contrast to its effect on bacterial systems, chloramphenicol has been reported to have little or no action on the protein synthesis by cell-free extracts of mammalian cells (Rendi, 1959; Ehrenstein & Lipmann, 1961). A basis for this resistance has been proposed by Vazquez (1964), who finds that whereas bacterial ribosomes bind chloramphenicol, ribosomes from other organisms do not. Nevertheless, it cannot be stated with any confidence that chloramphenicol has no effect on the protein synthesis of animal cells.


1972 ◽  
Vol 69 (5) ◽  
pp. 1299-1303 ◽  
Author(s):  
S. Metafora ◽  
M. Terada ◽  
L. W. Dow ◽  
P. A. Marks ◽  
A. Bank

1983 ◽  
Vol 29 (2) ◽  
pp. 181-195 ◽  
Author(s):  
Susan J. Kelly ◽  
Jean Loria M.T. Gyves ◽  
Judith Ilan

Sign in / Sign up

Export Citation Format

Share Document