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

Authors

Isao Masuda, Thomas Jefferson UniversityFollow
Takao Igarashi, Thomas Jefferson UniversityFollow
Reiko Sakaguchi, Thomas Jefferson UniversityFollow
Ram G. Nitharwal, Uppsala University
Ryuichi Takase, Thomas Jefferson UniversityFollow
Kyu Young Han, University of Illinois at Urbana-Champaign; University of Central Florida
Benjamin J. Leslie, University of Illinois at Urbana-Champaign; Howard Hughes Medical Institute
Cuiping Liu, Thomas Jefferson UniversityFollow
Howard Gamper, Thomas Jefferson UniversityFollow
Taekjip Ha, University of Illinois at Urbana-Champaign; Howard Hughes Medical Institute; Johns Hopkins University
Suparna Sanyal, Uppsala University
Ya-Ming Hou, Thomas Jefferson UniversityFollow

Document Type

Article

Publication Date

4-20-2017

Comments

This article has been peer reviewed. It is the author’s final published version in Nucleic Acids Research

Volume 45, Issue 7, April 2017, Pages 4081-4093

The published version is available at DOI: 10.1093/nar/gkw1229. Copyright © Masuda et al.

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.

Recommended Citation

Masuda, Isao; Igarashi, Takao; Sakaguchi, Reiko; Nitharwal, Ram G.; Takase, Ryuichi; Han, Kyu Young; Leslie, Benjamin J.; Liu, Cuiping; Gamper, Howard; Ha, Taekjip; Sanyal, Suparna; and Hou, Ya-Ming, "A genetically encoded fluorescent tRNA is active in live-cell protein synthesis." (2017). Department of Biochemistry and Molecular Biology Faculty Papers. Paper 119.
https://jdc.jefferson.edu/bmpfp/119

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 License.

PubMed ID

27956502