Evolving tRNA^Sec for Efficient Canonical Incorporation of Selenocysteine

Abstract

Bacterial selenocysteine incorporation occurs in response to opal stop codons and is dependent on the presence of a selenocysteine insertion sequence (SECIS) element, which recruits the selenocysteine specific elongation factor and tRNASec 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. Although the site specific incorporation of selenocysteine is of great interest for protein engineering, the sequence constraints imposed by the adjoining SECIS element severely limit its use. We have evolved an E. coli tRNASec that is compatible with the canonical translation machinery and can suppress amber stop codons to incorporate selenocysteine with high efficiency. This evolved tRNASec allows the production of new recombinant selenoproteins containing structural motifs such as selenyl-sulfhydryl and diselenide bonds.

Future of the Genetic Code

“…The opening up of selenoprotein biochemistry via unconstrained proteome-wide Sec encoding enables the replacement of disulfide bridges by diselenide bridges in proteins [34,35]. As a result, metabolic engineering can be carried out replacing glutathione (GSSG) as a cellular redox buffer by selenoglutathione (GSeSeG): otherwise selenoglutathione would destabilize disulfide bridges in proteins, for the E°’ of −407 mV for GSeSeG is much more negative than that of −256 mV for GSSG [86].…”

“…The adaptation of the Sec pathway to NCAA encoding is rendered difficult by the requirement for a selenocysteine insertion sequence (SECIS) motif, and the non-binding of Sec-tRNA(Sec) to EF-Tu. However, both of these hurdles have been removed through the development of an effective tRNA(Sec) that incorporated Sec into proteins via EF-Tu binding, thus converting the highly restricted occurrence of Sec only at SECIS-directed mRNA contexts to unconstrained insertion as an NCAA anywhere in the proteome [34,35]. This liberated Sec pathway therefore can be adapted to the incorporation of NCAAs as in the case of the Pyl pathway.…”

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

“…tRNA engineering can also improve the efficiency of an oaaRS•tRNA pair by optimizing orthogonality of the tRNA, its binding to the o-aaRS or elongation factor Tu, or the decoding strength of the targeted codon [24–29] (Box 1). Also, the role of heterologous tRNA post-transcriptional modifications is emerging as a considerable factor in improving o-tRNA proficiency [30–32].…”

Rewriting the Genetic Code

“…Although Sec naturally requires the dedicated elongation factor SelB (33), two separate tRNAs have been reengineered to incorporate Sec using the more conventional elongation factor EF-Tu (2, 44, 89, 139), which is not dependent on a Sec insertion sequence in the mRNA. Finally, it would be beneficial to develop orthogonal aaRS•tRNA pairs that can function across all domains, like the tRNA Pyl •PylRS pair (97).…”