A Genetically Encoded Fluorescent Trna Is Active In Live-Cell Protein Synthesis

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.

Publication Date

4-20-2017

Publication Title

Nucleic Acids Research

Volume

45

Issue

7

Number of Pages

4081-4093

Document Type

Article

Personal Identifier

scopus

DOI Link

https://doi.org/10.1093/nar/gkw1229

Socpus ID

85019151374 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/85019151374

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