Pyrrolysyl-tRNA Synthetase, an Aminoacyl-tRNA Synthetase for Genetic Code Expansion

Abstract

Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22nd genetically encoded amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNAPyl. This unique aaRS•tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme’s anticodon-blind specific recognition of the cognate tRNAPyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS.

Understanding the Genetic Code

“…UAG (amber), used in smaller amounts but almost invariant with respect to GC content, can also code for pyrrolysine, which is an active-site residue in some methyltransferases (45). This amino acid is found most frequently in archaea but occasionally in bacteria (46). Of the three stop codons, UGA (opal) is used for chain termination primarily in high-GC-content organisms, but the actual frequency depends also on the organismal type (44).…”

Pyrrolysyl-tRNA Synthetase with a Unique Architecture Enhances the Availability of Lysine Derivatives in Synthetic Genetic Codes

“…Pyrrolysyl-tRNA synthetase has been applied for expanding the repertoire of genetically encoded amino acids [ 1 , 2 , 3 , 4 ]. PylRS naturally occurs in archaea and certain bacteria species attaching pyrrolysine to tRNA Pyl to translate the UAG amber stop codon [ 1 , 5 ].…”

“…Lastly, the tRNA Pyl recognition by PylRS does not involve the anticodon moiety and tRNA Pyl can be engineered to read any other codons than UAG without impairing aminoacylation [ 7 , 8 , 9 ]. Thus, multiple PylRS variants were recently developed to genetically encode more than 100 synthetic amino acids [ 2 , 4 ] including α-hydroxy acids [ 10 , 11 , 12 , 13 ].…”

Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

“…Therefore, expanding the repertoire of amino acids in translation is useful for developing novel protein functions (reviewed in Wang et al, 2006;Liu and Schultz, 2010). ''Orthogonal'' pairs of an engineered aminoacyl-tRNA synthetase and tRNA, including bacterial and archaeal pairs of tyrosyl-tRNA synthetase (TyrRS) and tRNA Tyr (CUA) (Wang and Schultz, 2001;Wang et al, 2002;Chin et al, 2002Chin et al, , 2003Kiga et al, 2002;Sakamoto et al, 2002) and archaeal pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNA Pyl (CUA) for the 22nd amino acid, pyrrolysine (Blight et al, 2004;Polycarpo et al, 2004), have enabled the site-specific incorporation of non-natural amino acids into proteins in response to the amber (UAG) codon (reviewed in Wang et al, 2006;Liu and Schultz, 2010;Wan et al, 2014;Chin, 2014;Crnkovi c et al, 2016;Brabham and Fascione, 2017;Chin, 2017;Wang, 2017;Vargas-Rodriguez et al, 2018;Tharp et al, 2018). Chemically reactive functional groups, such as alkene, alkyne, azide, and diazirine groups, on non-natural amino acids allow the post-translational labeling of proteins with detection probes, polymers, drugs, and UV crosslinkers (reviewed in Chin, 2014a, 2014b;Elliott et al, 2014;Nguyen et al, 2018).…”