title: Site-specific incorporation of nonnatural residues during in vitro protein biosynthesis with semisynthetic aminoacyl-tRNAs.
abstract: A method is presented for the incorporation of nonnatural amino acids into proteins during in vitro cell-free translation. A combination of chemical synthesis and run-off transcription was employed to prepare a semisynthetic, nonhypermodified tRNA(Gly) nonsense suppressor acylated with L-3-[125I]iodotyrosine. The presence of this synthetic tRNA during in vitro translation of mRNA containing a nonsense suppression site (e.g., a UAG termination codon) results in the incorporation of the nonnatural amino acid L-3-iodotyrosine into the polypeptide exclusively at the position corresponding to that site. Incorporation of the nonnatural amino acid L-3-[125I]iodotyrosine into the model polypeptide was assessed by quantitative and unambiguous determination of suppression efficiency, read-through, and site specificity of incorporation. Minor modifications of the method employed in this initial experiment also allow the rapid analysis of unlabeled acylated tRNA analogues. Under optimum conditions, the unlabeled amino acid L-3-iodotyrosine was found to be incorporated with a suppression efficiency of 65%. Other nonnatural residues, including N-methylphenylalanine, D-phenylalanine, and phenyllactic acid, were tested in the assay under these same conditions. Suppression efficiencies for this series ranged from 0 to 72% depending on the structure of the residue incorporated. Several other aspects of this methodology, such as tRNA structure and context effects, are briefly discussed.
authors.affiliation: Department of Biological Chemistry, University of California, Irvine 92717.
authors.forename: J D
authors.initials: JD
authors.lastname: Bain
authors.forename: E S
authors.initials: ES
authors.lastname: Diala
authors.forename: C G
authors.initials: CG
authors.lastname: Glabe
authors.forename: D A
authors.initials: DA
authors.lastname: Wacker
authors.forename: M H
authors.initials: MH
authors.lastname: Lyttle
authors.forename: T A
authors.initials: TA
authors.lastname: Dix
authors.forename: A R
authors.initials: AR
authors.lastname: Chamberlin
mesh_terms: D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D007470:Monoiodotyrosine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000235:genetics*; D012353:RNA, Transfer, Gly / Q000737:chemistry; D011817:Rabbits; D012156:Reticulocytes / Q000737:chemistry; D013489:Suppression, Genetic
chemical_list: D012346:RNA, Transfer, Amino Acyl; D012353:RNA, Transfer, Gly; D007470:Monoiodotyrosine