Safe and easy in vitro evaluation of tmRNA-SmpB-mediated trans-translation from ESKAPE pathogenic bacteria
- Marion THEPAUT1,
- Rodrigo CAMPOS-SILVA2,
- Eva RENARD3,
- Frederique BARLOY-HUBLER1,
- Eric ENNIFAR3,
- Daniel BOUJARD1 and
- Reynald GILLET1,4
- 1 Univ. Rennes, CNRS, IGDR UMR 6290;
- 2 Faculdade de Farmacia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil;
- 3 CNRS UPR 9002, Institut de Biologie Moleculaire et Cellulaire, Universite de Strasbourg
- ↵* Corresponding author; email: reynald.gillet{at}univ-rennes1.fr
Abstract
In bacteria, trans-translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens.
Keywords
- Received April 14, 2021.
- Accepted July 27, 2021.
- Published by Cold Spring Harbor Laboratory Press for the RNA Society
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