A quantitative understanding of how system composition and molecular properties conspire to determine the fidelity of translation is lacking. Our strategy directs an orthogonal tRNA to directly compete against endogenous tRNAs to decode individual targeted codons in a GFP reporter. Sets of directed sense codon reassignment measurements allow the isolation of particular factors contributing to translational fidelity. In this work, we isolated the effect of tRNA concentration on translational fidelity by evaluating reassignment of the 15 least commonly employed E. coli sense codons. Eight of the rarely used codons are reassigned with greater than 20 % efficiency. Both tRNA abundance and codon demand moderately inversely correlate with reassignment efficiency. Furthermore, the reassignment of rarely used codons does not appear to confer a fitness advantage relative to reassignment of other codons. These direct competition experiments also map potential targets for genetic code expansion. The isoleucine AUA codon is particularly attractive for the incorporation of noncanonical amino acids, with a nonoptimized reassignment efficiency of nearly 70 %.
Keywords: genetic code expansion; missense errors; protein engineering; sense codon reassignment; synthetic biology.
© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.