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Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment

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dc.contributor.authorBiddle, Wil, author
dc.contributor.authorSchmitt, Margaret A., author
dc.contributor.authorFisk, John D., author
dc.contributor.authorOxford University Press, publisher
dc.date.accessioned2017-02-27T16:47:54Z
dc.date.available2017-02-27T16:47:54Z
dc.date.issued2016-10-23
dc.description.abstractBreaking 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: tRNAOpt. We suspected a modification of the tRNAOptAUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNAOptAUG is converted to inosine. We identified tRNAOptAUG anticodon 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.
dc.description.sponsorshipPublished with support from the Colorado State University Libraries Open Access Research and Scholarship Fund.
dc.format.mediumborn digital
dc.format.mediumarticles
dc.identifier.bibliographicCitationBiddle, Will, Margaret A. Schmitt, John D. Fisk, Modification of Orthogonal tRNAs: Unexpected Consequences for Sense Codon Reassignment, Nucleic Acids Research 44, no. 21 (2016): 10042-10050. http://doi.org/10.1093/nar/gkw948
dc.identifier.doihttps://dx.doi.org/10.1093/nar/gkw948
dc.identifier.urihttp://hdl.handle.net/10217/179924
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado State University. Libraries
dc.relation.ispartofOpen Access Research and Scholarship Fund (OARS)
dc.rights.licenseThis article is open access and distributed under the terms and conditions of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0).
dc.rights.urihttps://creativecommons.org/licenses/by-nc/4.0/
dc.subjectadenosine
dc.subjectamino acids
dc.subjectanticodon
dc.subjectcodon nucleotides
dc.subjectgenetic code
dc.subjecthistidine
dc.subjectinosine
dc.subjecttransfer RNA
dc.titleModification of orthogonal tRNAs: unexpected consequences for sense codon reassignment
dc.typeText

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