Nonorthogonal tRNA^cys_Amber for protein and nascent chain labeling

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-cysteinyltRNA 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.

Ribosome Processivity and Co-translational Protein Folding

“…Mutagenesis of the nucleotide U32 to C in the anticodon loop restored the rate of accommodation of Cys-tRNA Cys UCA U32C to same value as measured for Cys-tRNA Cys GCA. Our results are supported by a recent publication reporting translational activity of an amber suppressor tRNA Cys from Bacillus subtilis that naturally contains C32/A38 in an E. coli cell-free translation system (Koubek et al, 2015).…”