title: In Vivo Biosynthesis of a β-Amino Acid-Containing Protein.
abstract: It has recently been reported that ribosomes from erythromycin-resistant Escherichia coli strains, when isolated in S30 extracts and incubated with chemically mis-acylated tRNA, can incorporate certain β-amino acids into full length DHFR in vitro. Here we report that wild-type E. coli EF-Tu and phenylalanyl-tRNA synthetase collaborate with these mutant ribosomes and others to incorporate β(3)-Phe analogs into full length DHFR in vivo. E. coli harboring the most active mutant ribosomes are robust, with a doubling time only 14% longer than wild-type. These results reveal the unexpected tolerance of E. coli and its translation machinery to the β(3)-amino acid backbone and should embolden in vivo selections for orthogonal translational machinery components that incorporate diverse β-amino acids into proteins and peptides. E. coli harboring mutant ribosomes may possess the capacity to incorporate many non-natural, non-α-amino acids into proteins and other sequence-programmed polymeric materials.
authors.affiliation: Department of Chemistry, ‡Department of Molecular, Cellular, and Developmental Biology, and §Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06520-8107, United States.
authors.forename: Clarissa
authors.initials: C
authors.lastname: Melo Czekster
authors.affiliation: Department of Chemistry, ‡Department of Molecular, Cellular, and Developmental Biology, and §Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06520-8107, United States.
authors.forename: Wesley E
authors.initials: WE
authors.lastname: Robertson
authors.affiliation: Department of Chemistry, ‡Department of Molecular, Cellular, and Developmental Biology, and §Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06520-8107, United States.
authors.forename: Allison S
authors.initials: AS
authors.lastname: Walker
authors.affiliation: Department of Chemistry, ‡Department of Molecular, Cellular, and Developmental Biology, and §Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06520-8107, United States.
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.affiliation: Department of Chemistry, ‡Department of Molecular, Cellular, and Developmental Biology, and §Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06520-8107, United States.
authors.forename: Alanna
authors.initials: A
authors.lastname: Schepartz
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D012338:RNA, Ribosomal, 23S; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D012338:RNA, Ribosomal, 23S; D010649:Phenylalanine; D013762:Tetrahydrofolate Dehydrogenase; D010444:Peptide Elongation Factor Tu; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase