Reassembling green fluorescent protein for in vitro evaluation of trans-translation.
Guyomar C, Thépaut M, Nonin-Lecomte S, Méreau A, Goude R, Gillet R.
During translation, ribosomes translate the messenger RNA (mRNA) and synthesize the nascent poly-peptides until they encounter a stop codon on the mRNA. However, ribosomes regularly stall on the 3' end of mRNA, particularly when termination codon is missing. In bacteria, trans-translation is the main rescue mechanism for clearing these trapped ribosomes. This process is driven by transfer-messenger RNA, or tmRNA, along with its partner small protein B (SmpB). Trans-translation is an appealing target for new antibiotic molecules because: i) there is no trans-translation in eukaryotes, ii) it is essential for either the survival or virulence of many pathogenic bac-teria, and iii) in cases where the deletion is not lethal, it induces hypersensitive phenotypes thus making antibiotics more efficient.
In order to discover new antibiotics with improved activity and selectivity, researchers from IGDR have created a reliable in vitro reporter system to detect trans-translation activity. This system is based on an engineered tmRNA variant that reassembles the green fluorescent protein (GFP) when trans-translation is active. The system is adapted for high-throughput screening of chemical compounds by fluorescence.