An expanded genetic code with a functional quadruplet codon

Abstract

With few exceptions the genetic codes of all known organisms encode the same 20 amino acids, yet all that is required to add a new building block are a unique tRNA/aminoacyl-tRNA synthetase pair, a source of the amino acid, and a unique codon that specifies the amino acid. For example, the amber nonsense codon, TAG, together with orthogonal Methanococcus jannaschii or Escherichia coli tRNA/synthetase pairs have been used to genetically encode a variety of unnatural amino acids in E. coli and yeast, respectively. However, the availability of noncoding triplet codons ultimately limits the number of amino acids encoded by any organism. Here, we report the design and generation of an orthogonal synthetase/tRNA pair derived from archaeal tRNA(Lys) sequences that efficiently and selectively incorporates an unnatural amino acid into proteins in response to the quadruplet codon, AGGA. Frameshift suppression with L-homoglutamine (hGln) does not significantly affect protein yields or cell growth rates and is mutually orthogonal with amber suppression, permitting the simultaneous incorporation of two unnatural amino acids, hGln and O-methyl-L-tyrosine, at distinct positions within myoglobin. This work suggests that neither the number of available triplet codons nor the translational machinery itself represents a significant barrier to further expansion of the genetic code.

Fig. 1.

Construction of amber and four-base suppressor tRNAs. An amber suppressor tRNA was generated from sequence alignments of multiple archaeal tRNA^Lys sequences. The orthogonal AGGA suppressor tRNA was isolated by selection from an acceptor stem library.

Fig. 2.

(A) Structure of homoglutamine. (B) Schematic illustration of the PhKRS active site; residues Glu-41 and Tyr-268 make specific contacts with the lysine substrate (29). These two residues were simultaneously randomized.

Fig. 3.

Expression of mutant myoglobin by AGGA suppression. (A) Protein was produced from the myoglobin gene with an AGGA codon at position G24 (Myo24AGGA) only in the presence of all three components, AK514 tRNA, hGlnRS (an hGln-specific variant of PhKRSΔ), and hGln (lanes 2–5). Expression of myoglobin by amber suppression at position 75 (Myo75TAG) was only possible with JYRS and its cognate amber tRNA, demonstrating the mutual orthogonality of the lysyl and tyrosyl pairs (lanes 6–9). (B) Protein was expressed from a myoglobin gene containing AGGA at position 24 and TAG at position 75 (Myo24AGGA/75TAG) in the presence of both the lysyl and tyrosyl pairs. hGln was incorporated by AGGA suppression at position 24 and OMeTyr was incorporated by amber suppression at position 75 in a single polypeptide by using hGlnRS and OMeTyrRS. Protein was visualized with an anti-His6 antibody.

Fig. 4.

(A) Matrix-assisted laser desorption/ionization–time-of-flight analysis of tryptic fragments containing hGln (Upper) or Lys (Lower). (B) Electrospray MS analysis of full-length myoglobin containing hGln at position 24 and OMeTyr at position 75.