title: Performance of optimized noncanonical amino acid mutagenesis systems in the absence of release factor 1.
abstract: Site-specific incorporation of noncanonical amino acids (ncAAs) into proteins expressed in E. coli using UAG-suppression competes with termination mediated by release factor 1 (RF1). Recently, unconditional deletion of RF1 was achieved in a genomically recoded E. coli (C321), devoid of all endogenous UAG stop codons. Here we evaluate the efficiency of ncAA incorporation in this strain using optimized suppression vectors. Even though the absence of RF1 does not benefit the suppression efficiency of a single UAG codon, multi-site incorporation of a series of chemically distinct ncAAs was significantly improved.
authors.affiliation: Department of Chemistry, Boston College, 2609 Beacon Street, Chestnut Hill, MA 02467, USA. abhishek.chatterjee@bc.edu.
authors.forename: Yunan
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA and Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Lajoie
authors.affiliation: Department of Chemistry, Boston College, 2609 Beacon Street, Chestnut Hill, MA 02467, USA. abhishek.chatterjee@bc.edu.
authors.forename: James S
authors.initials: JS
authors.lastname: Italia
authors.affiliation: Department of Chemistry, Boston College, 2609 Beacon Street, Chestnut Hill, MA 02467, USA. abhishek.chatterjee@bc.edu.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Chin
authors.affiliation: Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
authors.affiliation: Department of Chemistry, Boston College, 2609 Beacon Street, Chestnut Hill, MA 02467, USA. abhishek.chatterjee@bc.edu.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Chatterjee
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015394:Molecular Structure; D016296:Mutagenesis; D010454:Peptide Termination Factors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; C489005:prfA protein, E coli