Environmental perturbations lift the degeneracy of the genetic code to regulate protein levels in bacteria

Abstract

The genetic code underlying protein synthesis is a canonical example of a degenerate biological system. Degeneracies in physical and biological systems can be lifted by external perturbations, thus allowing degenerate systems to exhibit a wide range of behaviors. Here we show that the degeneracy of the genetic code is lifted by environmental perturbations to regulate protein levels in living cells. By measuring protein synthesis rates from a synthetic reporter library in Escherichia coli, we find that environmental perturbations, such as reduction of cognate amino acid supply, lift the degeneracy of the genetic code by splitting codon families into a hierarchy of robust and sensitive synonymous codons. Rates of protein synthesis associated with robust codons are up to 100-fold higher than those associated with sensitive codons under these conditions. We find that the observed hierarchy between synonymous codons is not determined by usual rules associated with tRNA abundance and codon usage. Rather, competition among tRNA isoacceptors for aminoacylation underlies the robustness of protein synthesis. Remarkably, the hierarchy established using the synthetic library also explains the measured robustness of synthesis for endogenous proteins in E. coli. We further found that the same hierarchy is reflected in the fitness cost of synonymous mutations in amino acid biosynthesis genes and in the transcriptional control of σ-factor genes. Our study suggests that organisms can exploit degeneracy lifting as a general strategy to adapt protein synthesis to their environment.

Fig. 1.

Degeneracy lifting associated with amino acid limitation. (A) A library of 29 variants of the yellow fluorescent protein gene (yfp) was synthesized. In this library, each variant (represented as a horizontal line) was designed to measure the effect of one specific codon on protein synthesis rate. The identity of this codon and that of its cognate amino acid is indicated to the left of each yfp variant, and the locations of this codon along yfp are represented as thick vertical bars. Other codons for the same amino acid that were identical across all yfp variants in each codon family are represented as thin vertical bars. (B) Each yfp variant was constitutively expressed from a low-copy vector (SC101* ori, two copies per chromosome) in E. coli strains that were auxotrophic for one or more of seven amino acids. (C) To induce amino acid-limited growth, we adjusted the initial concentration of an amino acid in the growth medium to a level below that required for reaching saturating cell density. A methyl-ester analog of the amino acid supported steady growth in the amino acid-limited phase. Growth and fluorescence curves for two yfp variants, CTA, gray, and CTG, black, are shown as illustrative examples of degeneracy splitting upon limitation for the cognate amino acid, leucine. (D) Dark gray, YFP synthesis rates during limitation for cognate amino acid; light gray, YFP synthesis rates during amino acid-rich growth. YFP synthesis rate was defined as the rate of fluorescence change divided by the cell density. Synthesis rates were normalized by the maximum value within each synonymous codon family and separately in the amino acid-rich and amino acid-limited growth phases. Normalization factors (amino acid-rich, limited): Leu, 94, 81; Arg, 89, 113; Ser, 217, 343; Pro, 306, 49; Ile, 295, 45; Gln, 185, 83; Phe, 311, 20 (arbitrary units). Error bars show standard error over three replicate cultures.

Fig. 2.

Altering the hierarchy of degeneracy splitting among synonymous codons. The five leucine (arginine) tRNA isoacceptors were coexpressed together with each of the six leucine (arginine) yfp variants, resulting in 30 tRNA-yfp combinations for leucine (arginine). (A and B) Each square in the Left (Right) table corresponds to the difference in YFP synthesis rates of each yfp variant between the tRNA coexpressed strain and the parent strain without extra tRNA during leucine (arginine) limitation. YFP synthesis rates were defined in the same manner and normalized by the same factor as in Fig. 1D. (Left) YFP synthesis rate of the parent strain without extra tRNA during amino acid limitation is shown for each table (same data as in Fig. 1D). tRNA isoacceptor names are preceded by their unmodified anticodon sequences. Solid black-outlined squares correspond to codon–tRNA pairs that satisfy wobble-pairing rules after accounting for known posttranscriptional tRNA modifications (SI Appendix, Table S9). Dashed black-outlined squares correspond to codon–tRNA pairs that do not satisfy known wobble-pairing rules but that show a significant increase in YFP synthesis rate upon coexpression of the tRNA isoacceptor. ^UCGArg2_m is a nonnative arginine tRNA that was created by mutating the anticodon sequence of the ^ACGArg2 gene. Standard error was less than 0.05 for all squares. (C) Histogram of differences in YFP synthesis rate of yfp variants upon tRNA coexpression. Amino acid-limited growth, 42% median difference; amino acid-rich growth, 9% median difference (n = 60, aggregated for leucine and arginine). Change in YFP synthesis rate between each tRNA coexpressed strain and its parent strain expressing no extra tRNA was calculated as a percentage of the largest value between the two YFP synthesis rates.

Fig. 3.

Degeneracy lifting for endogenous proteins. (A) The effect of each codon on the synthesis rate, S, of a protein during amino acid limitation was modeled by a codon-specific weight, w_codon. The codon robustness index (CRI) for any protein-coding sequence was defined as the product of w_codon values for all codons in that sequence that are cognate to the limiting amino acid. (B) w_codon values for leucine and arginine codons during limitation for their cognate amino acids were estimated from protein synthesis rates of the corresponding yfp variants (Materials and Methods). w_codon values for all codons not cognate to the limiting acid were set to 1. (C) Ninety-two ORFs from the E. coli genome were cloned as N-terminal fusions to YFP downstream of a constitutive promoter into a low-copy vector (Inset and Materials and Methods). Robustness to leucine limitation is quantified as the ratio of protein synthesis rates between leucine-limited and leucine-rich growth phases. This measured robustness was correlated with estimated Leu CRI values for the 92 ORF-yfp fusions (r² = 0.61, squared Spearman’s rank correlation, P = 10⁻²⁰). Eleven ORFs had measured robustness below the lower limit of the vertical axis (SI Appendix, Table S1), but were included in the calculation of r². Protein synthesis rates were normalized by the synthesis rate for the CTG variant of yfp. Error bars show standard error over three replicate cultures. (D) Two sets of ORF-yfp fusions (21 total ORFs) were coexpressed with ^GAGLeu2 tRNA. (Left) On the basis of the yfp data (Fig. 2A), we estimated a higher CRI for the first set (11 ORFs) and a lower CRI for the second set (10 ORFs) upon ^GAGLeu2 coexpression (Materials and Methods). Hence we predicted that the first set should show an increase in robustness of protein synthesis during leucine limitation whereas the second set should show a decrease. (Right) These predictions agreed with measured changes for 20 of the 21 ORFs (r² = 0.57, P = 10⁻⁴). Error bars show standard error over three replicate cultures. Several error bars are smaller than data markers.

Fig. 4.

Fitness cost and transcriptional control reflect degeneracy lifting. (A) Four different prototrophic E. coli strains were created. Each of these strains had one of the four amino acid biosynthesis genes argA (Arg), carA (Arg), leuA (Leu), and leuC (Leu) replaced at the native locus by a corresponding synonymous mutant ORF. These mutants were designed such that three to five perturbation-robust codons in a wild-type ORF were replaced by perturbation-sensitive codons in the mutant ORF (SI Appendix, Fig. S12B). The strains were grown in medium supplemented with all 20 amino acids at 800 µM and then diluted into a medium lacking either leucine (Left) or arginine (Right). Growth lag was calculated as the time taken by each strain to reach OD₆₀₀ of 0.3 relative to a reference culture of the same strain grown in 800 µM of all 20 amino acids. (Left) Difference in growth lag between the leuA mutant and the two controls during leucine downshift was 9.2 ± 2.8 min, P = 10⁻³. (Right) Difference in growth lag between the carA mutant and the two controls during arginine downshift was 7.8 ± 1.2 min, P = 10⁻⁶. Standard errors were calculated over six biological replicates for each mutant. P values were calculated using a two-tailed t test between the leuA or the carA mutant and the corresponding controls. (B) (Upper) Genes encoding σ-factors and leucine biosynthesis genes in E. coli are biased in their Leu CRI values, as quantified using a z-score that measures the normalized deviation from the expected CRI value based on genome-wide codon frequencies (SI Appendix). The most frequent leucine codon CTG was excluded in this analysis because its frequency varies significantly with expression level under nutrient-rich conditions (38). (Lower) Fold change in mRNA abundance in response to leucine limitation for σ-factor genes and leucine biosynthesis operons was measured using RT-qPCR. Fold change of the gapA gene was used for internal normalization. Error bars show standard error over triplicate qPCR measurements.