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. Author manuscript; available in PMC: 2021 Aug 4.
Published in final edited form as: ACS Chem Biol. 2021 Mar 16;16(4):766–774. doi: 10.1021/acschembio.1c00120

Figure 2.

Figure 2.

Reassigning sense codons for initiation with ncAAs. (A) A series of sfGFP reporters was constructed in which the initiating methionine codon was replaced with UAG or one of eight sense codons. The ribosome binding site and initiating codon are shown in blue and bold text, respectively. (B) Co-expression of a mutant initiator tRNA enables translation initiation with ncAAs at select codons. Data are displayed as the mean ± SEM of three biological replicates (*p < 0.05, **p < 0.005, paired t test). (C) Relative expression of sfGFP initiating at AUG, UAG, and UAU. Data are displayed as the mean ± SEM of six biological replicates. (D) Expression of sfGFP[1UAU] is dependent on the addition of pMeF to the growth media. (E) LC-MS of panel D. Peaks corresponding to sfGFP-1pMeF (theoretical mass = 27786 Da) and N-formyl-sfGFP-1pMeF (theoretical mass = 27814 Da) were detected. (F) itRNATy2AUA is not aminoacylated by the E. coli TyrRS. Time points represent the mean of three independent experiments. Error bars are contained within each data point. (G) ncAA-dependent expression of codon-optimized sfGFP in which all elongating UAU codons were replaced with UAC.