Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins

Abstract

Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein’s function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of Nε-acetyl-l-lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid via mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications.

Suppressor tRNAs at the interface of genetic code expansion and medicine

“…For example, the natural suppressor tRNA Pyl is more efficient at translating its cognate codon UAG than UGA. It can also mistranslate UAG, albeit with two times lower efficiency (Morosky et al, 2023). Moreover, intrinsic tRNA elements contribute to codon specificity, as shown for a synthetic sup-tRNA for selenocysteine with CUA anticodon that mistranslates UGA codons with similar efficiency as the cognate UAG (Morosky et al, 2023).…”

“…It can also mistranslate UAG, albeit with two times lower efficiency (Morosky et al, 2023). Moreover, intrinsic tRNA elements contribute to codon specificity, as shown for a synthetic sup-tRNA for selenocysteine with CUA anticodon that mistranslates UGA codons with similar efficiency as the cognate UAG (Morosky et al, 2023). However, the same sup-tRNA with UCA anticodon is specific for UGA.…”

Orthogonal Translation for Site-Specific Installation of Post-translational Modifications

“…In the same work, another frequently used orthogonal pair, the Mj TyrRS/tRNA Tyr pair, was shown to be orthogonal to both pairs of Mb PylRS/tRNA Pyl and Mm SepRS/tRNA Sep , indicating the possibility of installing three distinct PTMs into a single protein. Using a similar strategy, acetyllysine and selenocysteine have been genetically encoded into proteins simultaneously. , Recently, several approaches to site-specifically incorporating three distinct ncAAs into a single protein have been reported. For example, OTSs based on Ec TyrRS, Ec TrpRS, and Mb PylRS are mutually orthogonal to each other and are able to decode UAG, UGA, and UAA codons as three distinct ncAAs in mammalian cells respectively, while OTSs derived from Ec LeuRS, Mm PylRS and Ec TyrRS are also mutually orthogonal to each other and can read UAG, UGA, and UAA codons as three different ncAAs in mammalian cells separately .…”

Tuning tRNAs for improved translation

“…Further engineering of the tRNA UTu1 D-arm to improve SelA binding showed a significant improvement in Sec insertion. This tRNA and others (allo-tRNA UTu2D ) have since been reliably used for downstream applications ( Evans et al, 2021 ; Evans et al, 2024 ; Morosky et al, 2023 ).…”