Genetic Code Engineering in the Study of Post-translational Modifications

The common genetic code is composed of 64 triplet codons that encode the incorporation of 20 canonical amino acids with a limited set of chemical groups. However, this chemical diversity can be modified by the use of an expanded genetic code that encodes for more than 20 amino acids. In this synthetic genetic code, one codon (or more) is recoded to encode a non-canonical amino acid. The co-translational and site-specific incorporation of this non-canonical amino acid is enabled by the use of orthogonal translation machinery components, namely, an orthogonal tRNA synthetase/tRNA pair. For example, the post translational modification of lysine residues by acetylation can be genetically encoded, by recoding the UAG stop codon to encode the incorporation of acetyl lysine. In recent years, thousands of potential lysine acylation sites have been identified, demonstrating that lysine acylation—mainly acetylation—is a widespread post-translational modification that occurs on proteins involved in diverse cellular functions. By genetically encoding the site-specific incorporation of acetyl lysine, we studied the role of acetylation in the regulation of glucose metabolism, using full-length and site-specifically acetylated enzymes expressed in cultured mammalian cells and bacteria. In addition, we genetically encode analogues of acetyl lysine that serve as suicide substrates of lysine deacetylases, in order to study the interactions between deacetylases and their full-length and site-specifically acetylated substrates. Our array of newly developed technologies should enable the study of virtually any site-specifically acetylated full-length protein.