Co‐translational Installation of Posttranslational Modifications by Non‐canonical Amino Acid Mutagenesis

Abstract

Protein posttranslational modifications (PTMs) play critical roles in regulating cellular activities. Here we provide a survey of genetic code expansion (GCE) methods that were applied in the co‐translational installation and studies of PTMs through noncanonical amino acid (ncAA) mutagenesis. We begin by reviewing types of PTM that have been installed by GCE with a focus on modifications of tyrosine, serine, threonine, lysine, and arginine residues. We also discuss examples of applying these methods in biological studies. Finally, we end the piece with a short discussion on the challenges and the opportunities of the field.

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

“…172 The applications of these AcK-OTSs in determining the roles of lysine acetylation in regulating specific proteins, from histones to nonhistone proteins, have been nicely reviewed recently. 36,38,40,173 There is one potential problem when using AcK-OTSs for in vivo studies. Due to existence of deacetylases, site-specifically incorporated acetyllysine residues by GCE undergo deacetylation, especially in yeast or mammalian cells, and the stoichiometry of acetylation at target sites varies from batch to batch to make inconsistence among replicates.…”

“…Furthermore, because of the orthogonality of PylRS-derived OTSs in both bacteria and eukaryotes, genetic encoding of acetyllysine into proteins has been established in different organisms, including E. coli , , Salmonella , yeast, , mammalian cells, , stem cells, and animals . The applications of these AcK-OTSs in determining the roles of lysine acetylation in regulating specific proteins, from histones to nonhistone proteins, have been nicely reviewed recently. ,,, …”

“…To fill in this gap, the genetic code expansion (GCE) strategy has been applied for PTM studies. − It introduces an orthogonal translation system (OTS) into living cells to genetically encode a noncanonical amino acid (ncAA) at a chosen site of target proteins. − The OTS is mainly composed of an engineered aminoacyl-tRNA synthetase (AARS), which recognizes the ncAA, and an engineered tRNA, which bears a specific anticodon to read an assigned codon in mRNA (Figure ). − The orthogonality of an OTS includes the following: First, the target ncAA cannot be recognized by host AARSs; Second, the engineered AARS cannot recognize canonical amino acids and cannot aminoacylate host tRNAs; Third, the engineered tRNA cannot be aminoacylated by host AARSs.…”

Noncanonical Amino Acids in Biocatalysis

“…In many cases, these covalent modifications cannot easily be recapitulated in recombinant proteins, and as a result their functional significance can be difficult to elucidate . GCE offers a powerful approach to probe the role of PTMs, through selective introduction of ncAAs that are structural mimics of post-translationally modified residues. − …”