title: Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.
abstract: We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.
authors.affiliation: Department of Biochemistry & Molecular Biology, Oregon Health & Sciences University , 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, United States.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hadd
authors.forename: John J
authors.initials: JJ
authors.lastname: Perona
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015202:Protein Engineering; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology
chemical_list: D001426:Bacterial Proteins; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase