biostudies-literatureThepaut Mthe EU&apos;s Joint Programming Initiative on Antimicrobial Resistance project (JPIAMR) project “Ribotarget—Development of novel ribosome-targeting antibiotics” as well as by SATT Ouest-ValorisationAgence Nationale pour la Recherche1390-1399https://www.ebi.ac.uk/biostudies/studies/S-EPMC8522692biostudies-literatureUnknown27(11)In bacteria, <i>trans</i>-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 <i>trans</i>-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 <i>trans</i>-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of <i>trans</i>-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active <i>trans</i>-translation, we have created a cell-free assay adapted to the rapid evaluation of <i>trans</i>-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 <i>trans</i>-translation in these pathogens.RNA (New York, N.Y.)Safe and easy in vitro evaluation of tmRNA-SmpB-mediated <i>trans</i>-translation from ESKAPE pathogenic bacteria.PMC852269225523506Ennifar ECampos-Silva RBarloy-Hubler FRenard EBoujard DGillet RThepaut MfalseSafe and easy in vitro evaluation of tmRNA-SmpB-mediated <i>trans</i>-translation from ESKAPE pathogenic bacteria.In bacteria, <i>trans</i>-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 <i>trans</i>-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 <i>trans</i>-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of <i>trans</i>-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active <i>trans</i>-translation, we have created a cell-free assay adapted to the rapid evaluation of <i>trans</i>-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 <i>trans</i>-translation in these pathogens.2021-01-01T00:00:00Z2021 Nov2025-04-05T13:42:43.673Z2025-04-05T13:42:43.673ZS-EPMC85226923435392510.1261/rna.078773.121