The rate of the molecular clock and the cost of gratuitous protein synthesis

Abstract

Background: The nature of the protein molecular clock, the protein-specific rate of amino acid substitutions, is among the central questions of molecular evolution. Protein expression level is the dominant determinant of the clock rate in a number of organisms. It has been suggested that highly expressed proteins evolve slowly in all species mainly to maintain robustness to translation errors that generate toxic misfolded proteins. Here we investigate this hypothesis experimentally by comparing the growth rate of Escherichia coli expressing wild type and misfolding-prone variants of the LacZ protein.

Results: We show that the cost of toxic protein misfolding is small compared to other costs associated with protein synthesis. Complementary computational analyses demonstrate that there is also a relatively weaker, but statistically significant, selection for increasing solubility and polarity in highly expressed E. coli proteins.

Conclusions: Although we cannot rule out the possibility that selection against misfolding toxicity significantly affects the protein clock in species other than E. coli, our results suggest that it is unlikely to be the dominant and universal factor determining the clock rate in all organisms. We find that in this bacterium other costs associated with protein synthesis are likely to play an important role. Interestingly, our experiments also suggest significant costs associated with volume effects, such as jamming of the cellular environment with unnecessary proteins.

Figure 1

Expression of destabilizing mutants and wild-type LacZ. (a) SDS-PAGE of soluble and insoluble fractions of cells expressing WT LacZ and five destabilizing mutants induced with 10 μM IPTG. (b) Total β-galactosidase at different times after IPTG induction. The LacZ band is indicated by the black arrow. (c) Relative synthesis rate of β-galactosidase. P-values were obtained using a t-test of the linear regression slopes based on quantification of the gel images. Error bars represent the standard error of the regression slopes. (d) GroEL western blots in cells exprerssing WT and LacZ mutants. S, soluble fraction; I, insoluble fraction; '-', no IPTG; '+', 20 μM IPTG; Δ, heat shock (1 h shift from 37 to 42°C).

Figure 2

Comparison of the growth rates for wild-type and misfolding-prone LacZ. (a) Growth rates of cells expressing WT LacZ relative to uninduced cells and cells expressing each of the five destabilizing mutants (10 μM IPTG). Mann-Whitney U P-value: *0.02; **8 × 10^-4. (b) Growth rates of cells expressing WT LacZ and two mutants at different induction (IPTG) levels; the growth rate of cells carrying an empty plasmid is also shown for comparison. (c) Growth rates of cells expressing LacZ and two destabilizing mutants on acetate and glycerol as the main carbon source; in both cases expression was induced with 10 μM ITPG). Error bars represent the standard error of the mean calculated based on triplicate experiments.

Figure 3

Correlation of E. coli mRNA expression with Ka, protein solubility, and the fraction of charged residues. (a) Correlation between expression and the rate of non-synonymous substitutions (Ka; Spearman's r = -0.45, P < 10^-10). (b) Correlation between Ka and the rate of synonymous substitutions (Ks; r = 0.66, P < 10^-10). (c) Correlation between expression and protein solubility measured in vitro [48] (r = 0.27, P < 10^-10). (d) Correlation between expression and the fraction of charged residues (r = 0.28, P < 10^-10). The red lines on each panel represent a 200-point moving average.

Figure 4

Relationship between protein stability and mRNA expression. The experimentally measured stability data were obtained from the ProTherm database [54], and the expression data for E. coli were obtained from the study by Lu et al. [78]. (a) Correlation between mRNA expression and melting temperature for 28 proteins (r = -0.14, P = 0.45). (b) Correlation between mRNA expression and folding free energy for 23 proteins (r = -0.08, P = 0.70). The dashed red line represents the linear regression between each variable and the natural logarithm of the expression values.