Characterization of an 'orthogonal' suppressor tRNA derived from E. coli tRNA2(Gln)

In an effort to expand further our ability to manipulate protein structure, we have completed the first step towards a general method that allows the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the construction of an 'orthogonal' suppressor tRNA that is uniquely acylated in vivo, by an engineered aminoacyl-tRNA synthetase, with the desired unnatural amino acid. The Escherichia coli tRNA2(Gln)-glutaminyl-tRNA synthetase (GlnRS) pair provides a biochemically and structurally well-characterized starting point for developing this methodology. To generate the orthogonal tRNA, mutations were introduced into the acceptor stem, D-loop/stem, and anticodon loop of tRNA2(Gln). We report here the characterization of the properties of the resulting tRNAs and their suitability to severe as an orthogonal suppressor. Our efforts to generate an engineered synthetase are described elsewhere.

Mutant tRNAs were generated by runoff transcription and assayed for their ability to be aminoacylated by purified E. coli GlnRS and to suppress an amber codon in an in vitro transcription/translation reaction. One tRNA bearing eight mutations satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase, including GlnRS, yet functions efficiently during protein translation. Mutations in the acceptor stem and D-loop/stem, when introduced in combination, had very different effects on the properties of the resulting tRNAs compared with the effects of the individual mutations.

Mutations at sites within tRNA2(Gln) separated by 23-31 A interact strongly with each other, often in a nonadditive fashion, to modulate both aminoacylation activities and translational efficiencies. The observed correlation between the effects of mutations at very distinct regions of the GlnRS-tRNA and possibly the ribosomal/tRNA complexes may contribute in part to the fidelity of protein biosynthesis.