A genetically encoded fluorescent tRNA is active in live-cell protein synthesis

Isao Masuda, Takao Igarashi, Reiko Sakaguchi, Ram G. Nitharwal, Ryuichi Takase, Kyu Young Han, Benjamin J. Leslie, Cuiping Liu, Howard Gamper, Taekjip Ha, Suparna Sanyal, Ya Ming Hou

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4081-4093
Number of pages13
JournalNucleic acids research
Volume45
Issue number7
DOIs
StatePublished - Apr 20 2017

ASJC Scopus subject areas

  • Genetics

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