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. 2017 Apr 20;45(7):3615-3626.
doi: 10.1093/nar/gkx070.

Measurements of Translation Initiation From All 64 Codons in E. Coli

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Free PMC article

Measurements of Translation Initiation From All 64 Codons in E. Coli

Ariel Hecht et al. Nucleic Acids Res. .
Free PMC article

Abstract

Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the 'start codons' for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.

Figures

Figure 1.
Figure 1.
Plasmid sets used to measure translation initiation from non-canonical start codons. Plasmids varied in origin of replication (copy number), promoter and reporter gene characteristics. (A) Set of 64 pET20b(+) plasmids containing medium-copy pBR322 origin, T7 promoter and GFP reporter. (B) Set of 12 plasmids containing low-copy p15A origin, RhaPBAD rhamnose-inducible native Escherichia coli promoter and GFP reporter. (C) Set of 12 plasmids containing low-copy p15A origin, RhaPBAD rhamnose-inducible native E. coli promoter and nanoluciferase reporter. (D) Set of 12 very-low-copy bacterial artificial chromosomes (BAC) containing RhaPBAD rhamnose-inducible native E. coli promoter and nanoluciferase reporter.
Figure 2.
Figure 2.
Translation initiation from all 64 codons. Normalized per-cell fluorescence measured from three replicate cultures, grown in LB and resuspended in PBS before measurement, with each of the 64 codons as the start codon in the GFP coding sequence. Shapes represent the replicate plate number, and filled shapes represent GFP expression significantly greater (adjusted P < 0.05) than the non-expressing control (Control) as determined by Dunnett's test.
Figure 3.
Figure 3.
Translation initiation from a subset of 12 codons spanning the expression range. Translation initiated from three expression cassettes, (A) GFP on a low-copy p15A plasmid, (B) nanoluciferase on a low-copy p15A plasmid and (C) nanoluciferase on a very-low-copy BAC. Transcription was driven by the RhaPBAD rhamnose-inducible native E. coli promoter. Shapes represent the replicate plate number, and filled shapes represent expression significantly greater (adjusted P < 0.05) than the non-expressing control (Control) as determined by Dunnett's test.

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