title: Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids.
abstract: Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNA(Pyl) to create tRNA(Pyl-opt) with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (N(ϵ)-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNA(Pyl-opt) facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNA(Pyl-opt) with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNA(Pyl-opt) had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNA(Pyl-opt) should be an excellent replacement of wild-type tRNA(Pyl) for future ncAA incorporation by PylRS enzymes.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8144, USA chenguang.fan@yale.edu.
authors.forename: Chenguang
authors.initials: C
authors.lastname: Fan
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8144, USA.
authors.forename: Hai
authors.initials: H
authors.lastname: Xiong
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8144, USA.
authors.forename: Noah M
authors.initials: NM
authors.lastname: Reynolds
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8144, USA Department of Chemistry, Yale University, New Haven, CT 06520-8144, USA dieter.soll@yale.edu.
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006657:Histones / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008291:Malate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D046249:Transfer RNA Aminoacylation
chemical_list: D000596:Amino Acids; D006657:Histones; D049452:Green Fluorescent Proteins; D008291:Malate Dehydrogenase; D000604:Amino Acyl-tRNA Synthetases