Nonorthogonal tRNA(cys)(Amber) for protein and nascent chain labeling

Jiří Koubek; Yet-Ran Chen; Richard Ping Cheng; Joseph Jen-Tse Huang

doi:10.1261/rna.051805.115

Nonorthogonal tRNA(cys)(Amber) for protein and nascent chain labeling

RNA. 2015 Sep;21(9):1672-82. doi: 10.1261/rna.051805.115. Epub 2015 Jul 20.

Authors

Jiří Koubek¹, Yet-Ran Chen², Richard Ping Cheng³, Joseph Jen-Tse Huang⁴

Affiliations

¹ Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Nankang, Taipei 11529, Taiwan Institute of Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
² Agricultural Biotechnology Research Center, Academia Sinica, Nankang, Taipei 11529, Taiwan.
³ Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
⁴ Institute of Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan.

Abstract

In vitro-transcribed suppressor tRNAs are commonly used in site-specific fluorescence labeling for protein and ribosome-bound nascent chains (RNCs) studies. Here, we describe the production of nonorthogonal Bacillus subtilis tRNA(cys)(Amber) from Escherichia coli, a process that is superior to in vitro transcription in terms of yield, ease of manipulation, and tRNA stability. As cysteinyl-tRNA synthetase was previously shown to aminoacylate tRNA(cys)(Amber) with lower efficiency, multiple tRNA synthetase mutants were designed to optimize aminoacylation. Aminoacylated tRNA was conjugated to a fluorophore to produce BODIPY FL-cysteinyl-tRNA(cys)(Amber), which was used to generate ribosome-bound nascent chains of different lengths with the fluorophore incorporated at various predetermined sites. This tRNA tool may be beneficial in the site-specific labeling of full-length proteins as well as RNCs for biophysical and biological research.

Keywords: aminoacylation; fluorescence labeling; ribosome-bound nascent chain; suppressor tRNA; time-resolved fluorescence anisotropy.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acyl-tRNA Synthetases / genetics
Amino Acyl-tRNA Synthetases / metabolism*
Bacillus subtilis / genetics*
Cell-Free System
Escherichia coli / genetics*
Escherichia coli / metabolism
Fluorescent Dyes / chemistry
In Vitro Techniques
Models, Molecular
Protein Biosynthesis
RNA Stability
RNA, Bacterial / biosynthesis
RNA, Bacterial / chemistry
RNA, Bacterial / genetics
RNA, Transfer, Cys / biosynthesis*
RNA, Transfer, Cys / chemistry*
RNA, Transfer, Cys / genetics
Transfer RNA Aminoacylation

Substances

Fluorescent Dyes
RNA, Bacterial
RNA, Transfer, Cys
Amino Acyl-tRNA Synthetases
cysteinyl-tRNA synthetase