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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. © 2015 Koubek et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.


Jiří Koubek, Yet-Ran Chen, Richard Ping Cheng, Joseph Jen-Tse Huang. Nonorthogonal tRNA(cys)(Amber) for protein and nascent chain labeling. RNA (New York, N.Y.). 2015 Sep;21(9):1672-82

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PMID: 26194135

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