Codon usage of highly expressed genes affects proteome-wide translation efficiency

Abstract

Although the genetic code is redundant, synonymous codons for the same amino acid are not used with equal frequencies in genomes, a phenomenon termed "codon usage bias." Previous studies have demonstrated that synonymous changes in a coding sequence can exert significant cis effects on the gene's expression level. However, whether the codon composition of a gene can also affect the translation efficiency of other genes has not been thoroughly explored. To study how codon usage bias influences the cellular economy of translation, we massively converted abundant codons to their rare synonymous counterpart in several highly expressed genes in Escherichia coli This perturbation reduces both the cellular fitness and the translation efficiency of genes that have high initiation rates and are naturally enriched with the manipulated codon, in agreement with theoretical predictions. Interestingly, we could alleviate the observed phenotypes by increasing the supply of the tRNA for the highly demanded codon, thus demonstrating that the codon usage of highly expressed genes was selected in evolution to maintain the efficiency of global protein translation.

Keywords: codon usage evolution; codon-to-tRNA balance; genome engineering; tRNA; translation efficiency.

Fig. 1.

Does the codon usage of a subset of genes affect the translation efficiency of other genes? (Upper) Hypothetical genomes of the wild-type and recoded strains are shown. Using genome engineering, we replaced abundant codons (origin codon, blue lines) with rare codons (destination codon, red lines) in highly expressed genes (white background). (Lower Left) Two potential effects of recoding on fitness: Recoding could either reduce or not affect the fitness. (Lower Center) The translation efficiency of recoded genes could be increased, decreased, or not changed. (Lower Right) The translation efficiency of non-recoded genes that have the origin (blue) or destination (red) codon could be increased, decreased, or not changed.

Fig. 2.

The Arginine CGN box. We recoded CGU and CGC (origin codons) to CGG (destination codon). In E. coli, both origin codons are translated by tRNA^ACG with the anticodon ACG due to an A-to-I modification that is mediated by the enzyme tRNA-specific adenosine deaminase (tadA). The destination codon is translated solely by tRNA^CCG, which translates no other codons. tRNA^ACG and tRNA^CCG appear in the genome with four copies and one copy, respectively. A solid arrow symbolizes fully matched interactions between the codon and anticodon; dashed arrows represent wobble interactions, which are enabled by modifying the ACG anticodon to ICG.

Fig. 3.

Manipulating the codon frequency of CGG results in global translation efficiency changes. (A) We carried out RNA-seq analysis of the transcriptome and mass spectrometry analysis of the proteome for both the wild-type and recoded strains. This allowed us to calculate the translation efficiency (protein/mRNA) for each gene and to classify two gene groups of increased or decreased translation efficiency with a fold-change threshold of 1.5. The eight recoded genes are colored black, the increased translation efficiency group is colored blue, the decreased translation efficiency group is colored red, and CGG-enriched genes are colored green. (Inset) Ratios of translation efficiency between recoded and wild-type cells for CGG-enriched genes (more than five occurrences of CGG) and CGG-depleted genes (no occurrences of CGG). CGG-enriched genes show lower translation efficiency ratios (P value = 0.01). (B) Distribution of CGG occurrences, translated by tRNA^CCG, for genes with increased (blue) or decreased (red) translation efficiency (TE) in the recoded strain compared with the wild-type strain. The group of genes with decreased translation efficiency demonstrates higher CGG occurrences (P value = 0.0018). (C) Distribution of CGU + CGC + CGA occurrences, all translated by tRNA^ACG, in genes with increased (blue) or decreased (red) translation efficiency in the recoded strain compared with the wild-type strain. The group of genes with increased translation efficiency demonstrates more codon CGU + CGC + CGA occurrences (P value = 6.79 × 10⁻⁵). (D) To increase the tRNA^CCG supply, we mutated the anticodon of tRNA^ACG from ACG to CCG on the background of the recoded strain and termed this strain the “anticodon-switched strain.” We then analyzed its transcriptome and proteome. Note that many fewer genes, and particularly the CGG-enriched genes in green, now deviate from the diagonal, suggesting that the anticodon-switching mutation alleviated the translational difficulty of the recoded strain. The color code is as in A. (Inset) CGG-enriched genes now show translation efficiency ratios similar to those of CGG-depleted genes (P value > 0.05). (E) As in B, but for the genes with increased and decreased translation efficiency in the anticodon-switched strain compared with the wild-type strain. In contrast to the previous comparison in B, these two groups utilize the CGG codon to the same extent (P value > 0.05). (F) As in C, but for the genes with increased and decreased translation efficiency between the wild-type and anticodon-switched strain. In contrast to the previous comparison in C, these two groups utilize the CGU + CGC + CGA codon to the same extent (P value > 0.05). (G) Translation initiation rates for genes with increased, decreased, or unaffected translation efficiency in the recoded and wild-type strains, as defined in A. Note that genes with decreased translation efficiency, which are also enriched with CGG, also show higher initiation rates (P value = 0.01), in agreement with theoretical predictions. (H) The translation efficacy pattern of the anticodon-switched (ACS) strain clustered closer to the wild-type strain and away from the recoded strain.

Fig. 4.

Codon manipulation affects proteomic codon usage. (A) We defined the codon proteomic usage as the number of codon occurrences in each gene multiplied by the measured expression level of its protein product. We calculated the recoded/wild type ratio of this index for each of the 61 sense codons and observed that the CGG codon has the lowest value. (B) As in A, but comparing the anticodon-switched strain with the wild-type strain. Due to the additional supply of tRNA^CCG, at the expense of tRNA^ACG, the CGG codon is among the highest codons, an indication that the additional tRNA supply improves the translation efficiency of genes containing CGG. Consistently, CGU and CGC show lower values than in A (see text for discussion on CGA codon).

Fig. 5.

Increased translational demand for CGG hampers the protein synthesis of a reporter gene. (A) To directly link frequency manipulation of CGG with the protein synthesis of other genes, we utilized two versions of a YFP-reporter gene with six occurrences of either CGU or CGG. These YFP reporters were introduced separately to either the wild-type or the recoded strain. Following the production of YFP vs. time along the growth cycle allowed us to derive the maximal YFP production for each combination of strain and YFP version. (B) For each strain, a YFP-CGG/YFP-CGU ratio is shown for maximal YFP production. The recoded strain demonstrates lower ratios for both these parameters compared with the wild-type strain (P value = 5.6 × 10⁻⁵), supporting our observation that changing the codon usage of a small subset of genes hampers the production of other genes that contain the CGG codon. Upon anticodon switching on the background of the recoded strain, the maximal YFP rate is restored to values similar to those in the wild-type strain.

Fig. 6.

A change in global translation efficiency patterns is deleterious. (A) Growth experiment (OD vs. time) of the wild-type strain (blue), the recoded strain (red), and the four anticodon-switched strains (tRNA^ACG argQ, dark orange; tRNA^ACG argZ, dark yellow; tRNA^ACG argY, bright yellow; and tRNA^ACG argV, bright orange). The recoded strain demonstrates a reduction in relative fitness to 0.87 compared with the wild-type strain (P value < 10⁻¹⁰). The four strains with anticodon switching (increased tRNA^CCG supply) on the background of the recoded strain demonstrate higher fitness compared with the recoded strain itself, demonstrating that restored translation efficiency patterns also alleviated the growth defect (relative fitness compared with recoded strain of switched argQ = 1.06, argZ = 1.08, argY = 1.02, and argV = 1.04). (B) Switching the anticodon of tRNA^ACG from ACG to CCG on the background of the wild-type strain reduces fitness (relative fitness of switched argQ = 0.95 and of argZ = 0.96 compared with the wild-type strain).

Fig. 7.

Introducing CGG on highly expressed genes results in mistranslation events. Using a methodology to identify translation errors from mass spectrometry data, we identified such events for two of the recoded genes, ompC and ompA, exactly at the positions at which CGU or CGC, respectively, was mutated into CGG. While we did not find errors in the wild-type strain, we did observe them in the recoded and anticodon-switched strains. The mistranslation event for ompC was found in the recoded strain, and it replaced the coded arginine with glutamine (which has a near-cognate anticodon, CUG) at position 238 of the protein. The mistranslation event for ompA was found in the anticodon-switched strain, and it replaced the coded arginine with lysine at position 329 of the protein.