title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
authors.forename: Isao
authors.initials: I
authors.lastname: Masuda
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
authors.forename: Takao
authors.initials: T
authors.lastname: Igarashi
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
authors.forename: Reiko
authors.initials: R
authors.lastname: Sakaguchi
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University, Box-596, BMC 75124, Uppsala, Sweden.
authors.forename: Ram G
authors.initials: RG
authors.lastname: Nitharwal
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Takase
authors.affiliation: Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.|CREOL, College of Optics & Photonics, University of Central Florida, 4304 Scorpius St., Orlando, FL 32816, USA.
authors.forename: Kyu Young
authors.initials: KY
authors.lastname: Han
authors.affiliation: Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.|Howard Hughes Medical Institute, Baltimore, MD 21205, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Leslie
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
authors.forename: Cuiping
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
authors.forename: Howard
authors.initials: H
authors.lastname: Gamper
authors.affiliation: Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.|Howard Hughes Medical Institute, Baltimore, MD 21205, USA.|Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.|Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA.|Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University, Box-596, BMC 75124, Uppsala, Sweden.
authors.forename: Suparna
authors.initials: S
authors.lastname: Sanyal
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
authors.forename: Ya-Ming
authors.initials: YM
authors.lastname: Hou
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D001593:Benzyl Compounds / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D048288:Imidazolines / Q000737:chemistry; D008856:Microscopy, Fluorescence; D014176:Protein Biosynthesis; D015347:RNA Probes / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D018724:Spinacia oleracea / Q000235:genetics
chemical_list: C560120:3,5-difluoro-4-hydroxybenzylidene imidazolinone; D052157:Aptamers, Nucleotide; D001593:Benzyl Compounds; D005456:Fluorescent Dyes; D048288:Imidazolines; D015347:RNA Probes; D012343:RNA, Transfer