Synonymous codon substitutions perturb cotranslational protein folding in vivo and impair cell fitness

Abstract

In the cell, proteins are synthesized from N to C terminus and begin to fold during translation. Cotranslational folding mechanisms are therefore linked to elongation rate, which varies as a function of synonymous codon usage. However, synonymous codon substitutions can affect many distinct cellular processes, which has complicated attempts to deconvolve the extent to which synonymous codon usage can promote or frustrate proper protein folding in vivo. Although previous studies have shown that some synonymous changes can lead to different final structures, other substitutions will likely be more subtle, perturbing predominantly the protein folding pathway without radically altering the final structure. Here we show that synonymous codon substitutions encoding a single essential enzyme lead to dramatically slower cell growth. These mutations do not prevent active enzyme formation; instead, they predominantly alter the protein folding mechanism, leading to enhanced degradation in vivo. These results support a model in which synonymous codon substitutions can impair cell fitness by significantly perturbing cotranslational protein folding mechanisms, despite the chaperoning provided by the cellular protein homeostasis network.

Keywords: cotranslational folding; elongation rate; protein design; ribosome; translation.

Fig. 1.

Chloramphenicol acetyltransferase (CAT) has a complex tertiary and quaternary structure. (A) Ribbon diagram depicting the native homotrimeric structure (Protein Data Bank ID: 3CLA) (34). (B) Schematic representation of the complex topology of the CAT monomer structure. Secondary structure elements are shown in rainbow order. Polka dots indicate the H β-strand in the central β-sheet contributed from an adjacent monomer. (C) Close up of the trimer interface, with the B and H β-strands in the central β-sheets colored as in B. Dashed lines indicate approximate monomer boundaries.

Fig. 2.

CAT encoded by the synonymous Shuf1 sequence leads to impaired E. coli growth in the presence of cam. (A) Relative codon usage in WT (black), Komar (11) (green), and Shuf1 (gray) CAT coding sequences. Positive values correspond to clusters of common codons, and negative values represent clusters of rare codons, calculated over a sliding window of 17 codons (37). (B) Growth curves of E. coli expressing ssrA-tagged CAT variants challenged with cam under low (200 ng/mL) or high (1,600 ng/mL) concentrations of inducer. (C) Relative abundance of untagged (solid bars) or ssrA-tagged (hatched bars) CAT accumulated in cells determined by quantitative Western blotting of cell lysates. (D) Growth curves in the absence of cam. In all figures, data points represent the mean ± SD of at least three independent experiments; **P < 0.01; ***P < 0.001; Welch’s t test.

Fig. 3.

Translation of CAT using Shuf1 coding sequence does not significantly perturb the E. coli proteome. Relative abundance of E. coli proteins upon expression of WT or Shuf1 CAT. Twelve E. coli molecular chaperones and AAA⁺ ATPases are shown in red; 1,264 other E. coli proteins are shown in black. No significant upregulation of chaperones or ATPases was observed for E. coli expressing Shuf1.

Fig. 4.

Proposed model for the effects of synonymous CAT codon substitutions on ssrA-tagged CAT folding and cell fitness. Synonymous changes in the Shuf1 coding sequence alter the local rate of translation, affecting the conformation of CAT cotranslationally and persisting after release of the nascent protein from the ribosome. These altered Shuf1 folding intermediates are more susceptible to degradation by ClpXP than intermediates populated during and after translation of the WT coding sequence. Some Shuf1-CATssrA proteins evade degradation and eventually fold to an active conformation that is also more susceptible to degradation than WT-CATssrA.

Fig. 5.

Shuf1 CAT is more susceptible to ClpXP degradation than WT CAT, despite several other indistinguishable characteristics. (A and B) Selective effects of ssrA-tagging and ClpX deletion on the Shuf1 growth defect. (A) In the ClpX deletion strain (W3110 ΔclpX), a large increase in growth rate relative to the parent strain is observed only for ssrA-tagged Shuf1. Other constructs grow slightly slower in the absence of ClpX. U, uninduced cell culture. (B) Cell growth data from A plotted to highlight the effect on growth rate of removing the ssrA tag. Omitting the ssrA tag has no effect on growth in the ClpX knockout (hatched bars). In the presence of ClpX (filled bars), there is a much larger increase in growth upon ssrA tag deletion for Shuf1 than WT, indicating Shuf1 is more susceptible to ClpXP degradation than WT. (C) Thermal denaturation of CAT monitored by far-UV CD spectroscopy at 205 nm. (D) Acetyltransferase activity of purified, native CAT, normalized to WT. (E) In vitro ClpXP degradation of native, purified, ssrA-tagged CAT trimers (43). In all panels, data points represent the mean ± SD; n = 3 biological replicates.