title: Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment.
abstract: Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNAOptWe suspected a modification of the tRNAOptAUGanticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNAOptAUGis converted to inosine. We identified tRNAOptAUGanticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms.
authors.affiliation: Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Wil
authors.initials: W
authors.lastname: Biddle
authors.affiliation: Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Schmitt
authors.affiliation: Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA nickfisk@colostate.edu.|Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523, USA.|School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: John D
authors.identifier: 0000-0001-9809-6140
authors.initials: JD
authors.lastname: Fisk
mesh_terms: D000241:Adenosine / Q000235:genetics / Q000378:metabolism; D000926:Anticodon; D003062:Codon; D019143:Evolution, Molecular; D005818:Genetic Engineering / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D007288:Inosine / Q000235:genetics / Q000378:metabolism; D017017:Methanococcus / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism*
chemical_list: D000926:Anticodon; D003062:Codon; D049452:Green Fluorescent Proteins; D006639:Histidine; D007288:Inosine; D012343:RNA, Transfer; D000241:Adenosine