Abstract
Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNAPhe on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNAfMet. By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNAPhe, underscoring the complexity of erythromycin’s effect on protein synthesis in bacterial cells.
Highlights
Ribosome-catalyzed protein synthesis is one of the most critical processes in any living cell
The effects of ERY on translation elongation depend on the nascent peptide sequence, where three alternative scenarios have been reported: peptidyl-tRNA drop-off,[4] nascent chain mediated translational arrest,[17,18,19] or peptide by-passing of the drug.[11,12,13]
In order to investigate the effects of ERY on ongoing translation elongation in live cells, we used a tRNA-tracking based approach to measure the rate of translation elongation directly
Summary
Ribosome-catalyzed protein synthesis is one of the most critical processes in any living cell. The NPET is a cavity about 100 A long and 10–20 A wide, spanning through the entire large 50S ribosomal subunit This tunnel allows the nascent peptide chain to pass through before getting released to the cytoplasm.[3]. Structural information on the binding site of ERY in the ribosome suggested that the drug allows translation only up to a specific peptide length, acting as a ‘plug-in-the-bottle.’. More evidence that ERY modulates translation, rather than blocking it,[2] comes from ribosome profiling experiments, which showed that even though the highest ribosome density locates at the beginning of genes, footprints occur
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
