title: 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-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.
authors.affiliation: 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.
authors.forename: Jiří
authors.initials: J
authors.lastname: Koubek
authors.affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Nankang, Taipei 11529, Taiwan.
authors.forename: Yet-Ran
authors.initials: YR
authors.lastname: Chen
authors.affiliation: Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Richard Ping
authors.initials: RP
authors.lastname: Cheng
authors.affiliation: Institute of Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan.
authors.forename: Joseph Jen-Tse
authors.initials: JJ
authors.lastname: Huang
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics*; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D066298:In Vitro Techniques; D008958:Models, Molecular; D014176:Protein Biosynthesis; D020871:RNA Stability; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012350:RNA, Transfer, Cys / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D046249:Transfer RNA Aminoacylation
chemical_list: D005456:Fluorescent Dyes; D012329:RNA, Bacterial; D012350:RNA, Transfer, Cys; D000604:Amino Acyl-tRNA Synthetases; C019975:cysteinyl-tRNA synthetase
keywords: aminoacylation; fluorescence labeling; ribosome-bound nascent chain; suppressor tRNA; time-resolved fluorescence anisotropy