Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation

Abstract

The incorporation of unnatural amino acids site-specifically is a valuable technique for structure-function studies, incorporation of biophysical probes, and determining protein-protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells.

Probing the role of the cation–π interaction in the binding sites of GPCRs using unnatural amino acids

“…As such, we cannot state with confidence what role this residue might play. We note that previous experiments have shown that the amino acid incorporated by misacylation of the THG73 tRNA is most likely Gln (45). If that is the case here, the mere 10-fold shift in EC 50 for what is effectively a W3.28Gln mutation is too small to be consistent with a cation-interaction.…”

“…If that is the case here, the mere 10-fold shift in EC 50 for what is effectively a W3.28Gln mutation is too small to be consistent with a cation-interaction. We also note that more recently developed tRNAs that are less prone to acylation may allow study of the W3.28 site (45,46).…”

Chemical Scale Studies of the Phe-Pro Conserved Motif in the Cys Loop of Cys Loop Receptors

“…THG73 (23) and TQOpSЈ (24,25) were used as amber suppressor tRNA and opal suppressor tRNA, respectively. Conjugated dCA-amino acid was ligated to 74-nucleotide tRNA as previously reported (21).…”

“…We investigated the possibility of a Phe-Pro interaction in this system by testing double mutant receptors in which Phe 135 was substituted with the non-aromatic Cha and Pro 136 was substituted with either Pip or 3-MePro, the two mutations that cause the largest EC 50 shifts. These experiments required consecutive incorporation of two different unnatural amino acids, an unprecedented experiment for receptors expressed in a living cell that was made possible by recent advances in tRNA design (24,25). The resulting current signals (1-4 A) were quite sufficient for quantitative analysis.…”

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

“…Like amber suppressors, opal and ochre tRNAs are usually derived from a native scaffold in which the anticodon and/or adjacent bases have been mutated to pair with a UGA or UAA codon, respectively (Köhrer et al 2004). tRNA Phederived opal and ochre tRNAs have been used to incorporate nitrophenylalanine in rabbit reticulocyte lysate (RRL) (Taira et al 2005), and opal suppressors derived from tRNA Gln or tRNA Trp have been used to incorporate 5-hydroxytryptophan (Zhang et al 2004) and 5-F-tryptophan (Rodriguez et al 2007), respectively, in mammalian cells. Ochre suppressors derived from Escherichia coli supF tRNA and the suppressors of all three nonsense codons derived from E. coli tRNA Gln have also been used to incorporate tyrosine (Köhrer et al 2003) and glutamine (Köhrer et al 2004) residues in mammalian cells, respectively.…”