A Genetic Tool to Quantify trans-Translation Activity in Vivo

In bacteria, trans-translation is the main quality control mechanism for rescuing ribosomes arrested during translation. This key process is universally conserved and plays a critical role in the viability and virulence of many pathogens. We developed a reliable in vivo double-fluorescence reporter...

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Bibliographic Details
Published in:Journal of molecular biology 2017-11, Vol.429 (23), p.3617-3625
Main Authors: Macé, Kevin, Demay, Fanny, Guyomar, Charlotte, Georgeault, Sylvie, Giudice, Emmanuel, Goude, Renan, Trautwetter, Annie, Ermel, Gwennola, Blanco, Carlos, Gillet, Reynald
Format: Article
Language:English
Subjects:
antibiotics
Bacteria - metabolism
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
fluorescence
Genetics
Life Sciences
Protein Biosynthesis
ribosome
Ribosomes - genetics
Ribosomes - metabolism
RNA, Bacterial - genetics
RNA, Bacterial - metabolism
RNA, Messenger - genetics
RNA, Messenger - metabolism
RNA-Binding Proteins - genetics
RNA-Binding Proteins - metabolism
screening
tmRNA
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Summary:In bacteria, trans-translation is the main quality control mechanism for rescuing ribosomes arrested during translation. This key process is universally conserved and plays a critical role in the viability and virulence of many pathogens. We developed a reliable in vivo double-fluorescence reporter system for the simultaneous quantification of both trans-translation and the associated proteolysis activities in bacteria. The assay was validated using mutant bacteria lacking tmRNA, SmpB, and the ClpP protease. Both antisense tmRNA-binding RNA and a peptide mimicking the SmpB C-terminal tail proved to be potent inhibitors of trans-translation in vivo. The double-fluorescent reporter was also tested with KKL-35, an oxadiazole derivative that is supposed to be a promising trans-translation inhibitor, and it surprisingly turns out that trans-translation is not the only target of KKL-35 in vivo. [Display omitted] •Novel reporter systems are needed for the rapid development of new antimicrobial drugs.•Inhibitors of trans-translation may act as antibiotics.•We developed and validated a double-fluorescence reporter system for simultaneous and specific detection of bacterial trans-translation and proteolysis activity.•Trans-translation is not the only target of KKL-35 in vivo.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2017.10.007