In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

Jacob Gubbens, Soo Jung Kim, Zhongying Yang, Arthur E. Johnson, William R. Skach

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNA Cys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent read through efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies.

Original languageEnglish (US)
Pages (from-to)1660-1672
Number of pages13
Issue number8
StatePublished - Aug 2010


  • Cell-free translation
  • Cysteine modification
  • Nonnatural amino acids
  • Suppressor tRNA
  • Translation termination
  • tRNA aminoacylation

ASJC Scopus subject areas

  • Molecular Biology


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