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Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC
Published on: March 15, 2013
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A genetically encoded fluorescent tRNA is active in live-cell protein synthesis
Isao Masuda1, Takao Igarashi1, Reiko Sakaguchi1
1Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
Nucleic Acids Research
|December 14, 2016
Summary
Researchers developed a fluorescent transfer RNA (tRNA) fusion probe for live imaging in E. coli. This novel probe monitors tRNA stability and is active in protein synthesis, offering new tools for cellular research.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Transfer RNAs (tRNAs) are crucial molecules involved in protein synthesis and cellular regulation.
- Existing methods for studying tRNA in live cells have limitations.
- Developing new imaging tools for tRNA is essential for understanding cellular processes.
Purpose of the Study:
- To develop a genetically encoded fluorescent tRNA fusion probe for live imaging in Escherichia coli.
- To assess the probe's utility in monitoring tRNA stability and its activity in protein synthesis.
- To explore the potential of this methodology for broader live-cell imaging applications.
Main Methods:
- Genetically encoding a fluorescent tRNA fusion incorporating a Spinach aptamer.
- Utilizing a cell-permeable, non-toxic fluorophore for fluorescence detection.
- Assessing tRNA stability via cellular quality control mechanisms.
- Evaluating peptidyl transfer activity and ribosome accommodation in live E. coli.
- Performing live-cell imaging to analyze probe localization relative to ribosomes and the nucleoid.
Main Results:
- The developed tRNA fusion probe is fluorescent upon fluorophore binding.
- The fusion probe is a viable tool for monitoring tRNA stability in a cellular quality control pathway.
- The probe is active in E. coli live-cell protein synthesis, supporting cell growth.
- Imaging revealed co-localization of the fusion probe and ribosomes in the cytosol, excluding the nucleoid.
Conclusions:
- The fluorescent tRNA fusion probe is a functional tool for live imaging of tRNA in E. coli.
- This methodology enables monitoring of tRNA stability and activity within living cells.
- The approach offers potential for developing advanced live-cell imaging tools with stoichiometric labeling capabilities for various cell types.
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