A Genetic Tool to Quantify trans-Translation Activity in Vivo

J Mol Biol. 2017 Nov 24;429(23):3617-3625. doi: 10.1016/j.jmb.2017.10.007. Epub 2017 Oct 13.

Abstract

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.

Keywords: antibiotics; fluorescence; ribosome; screening; tmRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteria / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Protein Biosynthesis*
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Ribosomes / genetics
  • Ribosomes / metabolism*

Substances

  • Bacterial Proteins
  • RNA, Bacterial
  • RNA, Messenger
  • RNA-Binding Proteins