title: Reassigning Sense Codon AGA to Encode Noncanonical Amino Acids in Escherichia coli.
abstract: A new method has been developed to reassign the rare codon AGA in Escherichia coli by engineering an orthogonal tRNA/aminoacyl-tRNA synthetase pair derived from Methanocaldococcus jannaschii. The tRNA mutant was introduced with a UCU anticodon, and the synthetase was evolved to correctly recognize the modified tRNA anticodon loop and to selectively charge a target noncanonical amino acid (NAA) onto the tRNA. In order to maximize the efficiency of AGA codon reassignment, while avoiding the lethal effects caused by global codon reassignment in cellular proteins, an inducible promoter (araBAD) was utilized to provide temporal controls for overexpression of the aminoacyl-tRNA synthetase and switch on codon reassignment. Using this system, we were able to efficiently incorporate p-acetylphenylalanine, O-methyl-tyrosine, and p-iodophenylalanine into proteins in response to AGA codons. Also, we found that E. coli strain GM10 was optimal in achieving the highest AGA reassignment rates. The successful reassignment of AGA codons reported here provides a new avenue to further expand the genetic code.
authors.affiliation: School of Natural Sciences, University of California, 5200 North Lake Road, Merced, CA, 95343, USA.
authors.forename: Yiyan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: School of Natural Sciences, University of California, 5200 North Lake Road, Merced, CA, 95343, USA.
authors.forename: Meng-Lin
authors.initials: ML
authors.lastname: Tsao
mesh_terms: D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism; D003062:Codon / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005815:Genetic Code; D063747:Methanocaldococcus / Q000201:enzymology; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism
chemical_list: D000596:Amino Acids; D003062:Codon; D014448:Tyrosine-tRNA Ligase
keywords: AGA; amino acid; aminoacyl-tRNA synthetase; mass spectrometry; tRNA