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Genes 2018, 9(11), 546; https://doi.org/10.3390/genes9110546

Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair

Department of Chemistry, University of Colorado Denver, Campus Box 194, P.O. Box 173364, Denver, CO 80217-3364, USA
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Received: 18 October 2018 / Revised: 5 November 2018 / Accepted: 5 November 2018 / Published: 12 November 2018
(This article belongs to the Special Issue Synthetic DNA and RNA Programming)
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Abstract

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1. View Full-Text
Keywords: genetic code expansion; release factor 1; amber stop codon suppression; M. jannaschii orthogonal pair; fluorescence-based screen genetic code expansion; release factor 1; amber stop codon suppression; M. jannaschii orthogonal pair; fluorescence-based screen
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Schwark, D.G.; Schmitt, M.A.; Fisk, J.D. Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair. Genes 2018, 9, 546.

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