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.
Highlights
To a first approximation, the process of translation involves the direct reading of three nucleotide codons in mRNA by three nucleotide anticodons of tRNAs employing standard Watson-Crick base-pairing rules
In order to better understand the factors controlling the overall efficiency of amber stop codon suppression by the orthogonal M. jannaschii tyrosyl tRNA/aminoacyl tRNA synthetase (aaRS) pair, we employed a fluorescence-based screen to quantify amber stop codon reassignment efficiencies at eight different tyrosine positions in superfolder green fluorescent protein in two E. coli strains
To remove complicating effects resulting from differences in protein folding and performance between mutants and control proteins, the suppression of amber stop codons was accomplished with the tyrosine-incorporating orthogonal M. jannaschii tRNA/aaRS pair, and stop codons were placed at tyrosine positions in each
Summary
The process of translation involves the direct reading of three nucleotide codons in mRNA by three nucleotide anticodons of tRNAs employing standard Watson-Crick base-pairing rules. This simple picture of translation is incomplete. Molecular modifications, and sequence context effects contribute to fine tune the efficiency of decoding individual codons in different mRNA contexts. System composition includes factors such as the relative concentrations of cognate and near cognate tRNAs, and in the case of non-sense suppression, suppressor tRNAs and release factors. The efficiency of competition is further modulated by tRNA modifications that impact tRNA–protein interactions as well as alter the energy of codon–anticodon pairings.
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