title: Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.
abstract: Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.
authors.affiliation: Department of Genetics, Harvard Medical School , Boston, Massachusetts 02115, United States.
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Ho
authors.forename: Noah M
authors.initials: NM
authors.lastname: Reynolds
authors.affiliation: Cold Spring Harbor Laboratory , Cold Spring Harbor, New York 11724, United States.
authors.forename: Keith
authors.initials: K
authors.lastname: Rivera
authors.affiliation: Cold Spring Harbor Laboratory , Cold Spring Harbor, New York 11724, United States.
authors.forename: Morgan
authors.initials: M
authors.lastname: Connolly
authors.forename: Li-Tao
authors.initials: LT
authors.lastname: Guo
authors.forename: Jiqiang
authors.initials: J
authors.lastname: Ling
authors.affiliation: Cold Spring Harbor Laboratory , Cold Spring Harbor, New York 11724, United States.
authors.forename: Darryl J
authors.initials: DJ
authors.lastname: Pappin
authors.affiliation: Department of Genetics, Harvard Medical School , Boston, Massachusetts 02115, United States.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
mesh_terms: D003062:Codon / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D012362:RNA, Transfer, Ser / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*
chemical_list: D003062:Codon; D012362:RNA, Transfer, Ser; D012694:Serine
keywords: 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS); aminoacyl-tRNA synthetase (aaRS); genetic code; noncanonical amino acid (ncAA); pyrrolysyl-tRNA synthetase (PylRS); quantitative proteomic analysis; selected reaction monitoring (SRM); sense codons reassignment; tRNA competition