Safe and easy in vitro evaluation of tmRNA-SmpB-mediated trans-translation from ESKAPE pathogenic bacteria
- Marion Thépaut1,2,5,
- Rodrigo Campos-Silva1,3,5,
- Eva Renard4,
- Frédérique Barloy-Hubler1,
- Eric Ennifar4,
- Daniel Boujard1 and
- Reynald Gillet1
- 1Université Rennes, CNRS, Institut de Génétique et Développement de Rennes (IGDR) UMR 6290, 35043 Rennes, France
- 2SATT Ouest-Valorisation, 35750 Rennes, France
- 3Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre 90610-000, Brazil
- 4Architecture et Réactivité de l'ARN—CNRS UPR 9002, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, 67084 Strasbourg, France
- Corresponding author: reynald.gillet{at}univ-rennes1.fr
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↵5 These authors contributed equally to this work.
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.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.078773.121.
- Received April 14, 2021.
- Accepted July 27, 2021.
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