title: Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo.
abstract: In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an "orthogonal" suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)-tRNA2Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase-tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein-tRNA interface and at sites further away, is discussed.
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, CA 94720, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Liu
authors.forename: T J
authors.initials: TJ
authors.lastname: Magliery
authors.forename: M
authors.initials: M
authors.lastname: Pastrnak
authors.forename: P G
authors.initials: PG
authors.lastname: Schultz
mesh_terms: D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005975:Glutamate-tRNA Ligase / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed
chemical_list: D005975:Glutamate-tRNA Ligase