Unleashing the 21st Amino Acid: Removing the Sequence Constraints of Selenocysteine Incorporation

Selenocysteine is a rare, naturally occurring amino acid with a mosaic distribution across all domains of life. Unlike canonical amino acids, selenocysteine does not exist freely, and is directly synthesized on its tRNA which is pre-charged with serine. In bacteria, selenocysteine is co-translationally incorporated in response to specific opal stop codons, designated by the presence of a selenocysteine insertion sequence (SECIS) element which recruits the selenocysteine specific elongation factor and charged tRNA^Sec needed to reassign the UGA codon. The SECIS element is a stem-loop RNA structure immediately following the UGA codon and forms part of the coding sequence in bacterial selenoproteins. With a low pKa and strong nucleophilicity,  the site specific incorporation of selenocysteine has great potential for protein engineering, but the sequence constraints imposed by the adjoining SECIS element severely limit its use. A potential solution to this problem is to interface selenocysteine incorporation with the canonical translation machinery. We developed a novel genetic reporter system which depends on the formation of an essential selenyl-sulfhydryl bond to confer antibiotic resistance, allowing high-throughput screening of tRNA^Sec variants compatible with the canonical elongation factor EF-Tu. From a tRNA^Sec library containing a variable antideterminant region, which blocks interaction with EF-Tu, we isolated a tRNA^Sec variant which is compatible with the canonical translation machinery and can suppress amber stop codons to incorporate selenocysteine with high efficiency. Using this evolved tRNA^Sec we have produced new recombinant selenoproteins containing structural motifs such as selenyl-sulfhydryl and diselenide bonds.