Coding-sequence determinants of gene expression in Escherichia coli

Abstract

Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate how, we engineered a synthetic library of 154 genes that varied randomly at synonymous sites, but all encoded the same green fluorescent protein (GFP). When expressed in Escherichia coli, GFP protein levels varied 250-fold across the library. GFP messenger RNA (mRNA) levels, mRNA degradation patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near the ribosomal binding site explained more than half the variation in protein levels. In our analysis, mRNA folding and associated rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.

FIGURE 1. Synthetic library of GFP genes with randomized codon usage

(A) Degenerate oligonucleotides were mixed and assembled by PCR. Fragments were then cloned, sequenced, and assembled into complete GFP genes. Red indicates third-codon positions. Degenerate symbols are as follows: D (A or G or T); H (A or C or T); N (A or C or G or T); R (A or G); Y (C or T). (B) Example alignment illustrating sequence diversity among fifteen synthetic genes. Shadowed boxes indicate first and second codon positions, which are conserved across the library. (C, D) The distribution of GC3 and CAI among the 154 synthetic GFP genes (C) is representative of the diversity among the 4,288 endogenous E. coli genes (D).

FIGURE 2. The determinants of gene expression

(A) Codon adaptation was not significantly correlated with fluorescence among the 154 GFP constructs (r=0.14, p=0.09). (B) Predicted 5’ mRNA folding energy was strongly correlated with fluorescence (r=0.66, p<1E-15). For each construct, folding energy was calculated in a window spanning positions −4 to +37 relative to translation start; two example structures are shown. (C) Sliding window analysis of mRNA folding and fluorescence. Local mRNA folding energies were calculated in a sliding window of length 42 nt. The significance of the correlation between local folding energy and fluorescence (negative log10 p-value) is plotted as a function of window position along the sequence. Note the overlapping locations of the 30-nt ribosomal binding site (blue bar) and the window of strongest correlation between folding energy and fluorescence (red bar, nt −4 through nt +37).

FIGURE 3. Expression levels of alternative GFP constructs

The distribution of log2 normalized fluorescence levels for pGK8 (T7 promoter, no leader sequence, top panel), pGK14 (P_BAD bacterial promoter, no leader sequence, middle panel) and pGK16 (trp/lac bacterial promoter, 28-codon leader sequence, bottom panel) expression vectors. Fluorescence varied substantially when expressed using T7 or bacterial promoter. The addition of a 28-codon leader sequence with low secondary structure produced uniformly high expression levels.