title: 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 bothin vitroandin vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In theMethanosarcinaceaethe incorporation of the 22ndgenetically 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 tRNAPylmake 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.
authors.affiliation: Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: Ana
authors.initials: A
authors.lastname: Crnković
authors.affiliation: Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: Tateki
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06520-8114, USA.; Department of Chemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.affiliation: Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: Noah M
authors.initials: NM
authors.lastname: Reynolds
keywords: genetic code expansion; non-canonical amino acid; pyrrolysyl-tRNA synthetase; stop codon suppression; synthetic biology; tRNAPyl