Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli

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Nucleic Acids Research. 2014, 42(12), pp. e100-e100. ISSN 0305-1048. eISSN 1362-4962. Available under: doi: 10.1093/nar/gku381
Zusammenfassung

While the structure of mature ribosomes is analyzed in atomic detail considerably less is known about their assembly process in living cells. This is mainly due to technical and conceptual hurdles. To analyze ribosome assembly in vivo, we designed and engineered an Escherichiacoli strain—using chromosomal gene knock-in techniques—that harbors large and small ribosomal subunits labeled with the fluorescent proteins EGFP and mCherry, respectively. A thorough characterization of this reporter strain revealed that its growth properties and translation apparatus were wild-type like. Alterations in the ratio of EGFP over mCherry fluorescence are supposed to indicate ribosome assembly defects. To provide proof of principle, subunit specific assembly defects were provoked and could be identified by both manual and fully automated fluorometric in vivo assays. This is to our knowledge the first methodology that directly detects ribosome assembly defects in vivo in a high-throughput compatible format. Screening of knock-out collections and small molecule libraries will allow identification of new ribosome assembly factors and possible inhibitors.

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ISO 690NIKOLAY, Rainer, Renate SCHLÖMER, Sabine SCHMIDT, Silke MÜLLER, Anja HEUBACH, Elke DEUERLING, 2014. Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli. In: Nucleic Acids Research. 2014, 42(12), pp. e100-e100. ISSN 0305-1048. eISSN 1362-4962. Available under: doi: 10.1093/nar/gku381
BibTex
@article{Nikolay2014-07Valid-28473,
  year={2014},
  doi={10.1093/nar/gku381},
  title={Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli},
  number={12},
  volume={42},
  issn={0305-1048},
  journal={Nucleic Acids Research},
  pages={e100--e100},
  author={Nikolay, Rainer and Schlömer, Renate and Schmidt, Sabine and Müller, Silke and Heubach, Anja and Deuerling, Elke}
}
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