title: Evaluating Sense Codon Reassignment with a Simple Fluorescence Screen.
abstract: Understanding the interactions that drive the fidelity of the genetic code and the limits to which modifications can be made without breaking the translational system has practical implications for understanding the molecular mechanisms of evolution as well as expanding the set of encodable amino acids, particularly those with chemistries not provided by Nature. Because 61 sense codons encode 20 amino acids, reassigning the meaning of sense codons provides an avenue for biosynthetic modification of proteins, furthering both fundamental and applied biochemical research. We developed a simple screen that exploits the absolute requirement for fluorescence of an active site tyrosine in green fluorescent protein (GFP) to probe the pliability of the degeneracy of the genetic code. Our screen monitors the restoration of the fluorophore of GFP by incorporation of a tyrosine in response to a sense codon typically assigned another meaning in the genetic code. We evaluated sense codon reassignment at four of the 21 sense codons read through wobble interactions in Escherichia coli using the Methanocaldococcus jannaschii orthogonal tRNA/aminoacyl tRNA synthetase pair originally developed and commonly used for amber stop codon suppression. By changing only the anticodon of the orthogonal tRNA, we achieved sense codon reassignment efficiencies between 1% (Phe UUU) and 6% (Lys AAG). Each of the orthogonal tRNAs preferentially decoded the codon traditionally read via a wobble interaction in E. coli with the exception of the orthogonal tRNA with an AUG anticodon, which incorporated tyrosine in response to both the His CAU and His CAC codons with approximately equal frequencies. We applied our screen in a high-throughput manner to evaluate a 10(9)-member combined tRNA/aminoacyl tRNA synthetase library to identify improved sense codon reassigning variants for the Lys AAG codon. A single rapid screen with the ability to broadly evaluate reassignable codons will facilitate identification and improvement of the combinations of sense codons and orthogonal pairs that display efficient reassignment.
authors.affiliation: Department of Chemistry, Colorado State University , Fort Collins, Colorado 80523, United States.
authors.forename: Wil
authors.initials: W
authors.lastname: Biddle
authors.affiliation: Department of Chemical and Biological Engineering, Colorado State University , Fort Collins, Colorado 80523, United States.
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Schmitt
authors.affiliation: Department of Chemical and Biological Engineering, Colorado State University , Fort Collins, Colorado 80523, United States.|Department of Chemistry, Colorado State University , Fort Collins, Colorado 80523, United States.|School of Biomedical Engineering, Colorado State University , Fort Collins, Colorado 80523, United States.
authors.forename: John D
authors.initials: JD
authors.lastname: Fisk
mesh_terms: D000926:Anticodon; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry; D063747:Methanocaldococcus / Q000235:genetics; D008958:Models, Molecular; D012343:RNA, Transfer / Q000235:genetics
chemical_list: D000926:Anticodon; D003062:Codon; D049452:Green Fluorescent Proteins; D012343:RNA, Transfer