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A genetically encoded fluorescent tRNA is active in live-cell protein synthesis
Thomas Jefferson Univ, Dept Biochem & Mol Biol, 233 South 10th St, Philadelphia, PA 19107 USA..
Thomas Jefferson Univ, Dept Biochem & Mol Biol, 233 South 10th St, Philadelphia, PA 19107 USA..
Thomas Jefferson Univ, Dept Biochem & Mol Biol, 233 South 10th St, Philadelphia, PA 19107 USA.;Kyoto Univ, Kyoto 6068501, Japan..
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2017 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 7, p. 4081-4093Article in journal (Refereed) Published
Abstract [en]

Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS , 2017. Vol. 45, no 7, p. 4081-4093
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Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-321801DOI: 10.1093/nar/gkw1229ISI: 000399448400047PubMedID: 27956502OAI: oai:DiVA.org:uu-321801DiVA, id: diva2:1094773
Funder
NIH (National Institute of Health), GM112659 GM114343 GM108972
Note

The two first authors contributed equally to this work.

Available from: 2017-05-11 Created: 2017-05-11 Last updated: 2017-05-11Bibliographically approved

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