Spurious regulatory connections dictate the expression‐fitness landscape of translation factors

During steady‐state cell growth, individual enzymatic fluxes can be directly inferred from growth rate by mass conservation, but the inverse problem remains unsolved. Perturbing the flux and expression of a single enzyme could have pleiotropic effects that may or may not dominate the impact on cell fitness. Here, we quantitatively dissect the molecular and global responses to varied expression of translation termination factors (peptide release factors, RFs) in the bacterium Bacillus subtilis . While endogenous RF expression maximizes proliferation, deviations in expression lead to unexpected distal regulatory responses that dictate fitness reduction. Molecularly, RF depletion causes expression imbalance at specific operons, which activates master regulators and detrimentally overrides the transcriptome. Through these spurious connections, RF abundances are thus entrenched by focal points within the regulatory network, in one case located at a single stop codon. Such regulatory entrenchment suggests that predictive bottom‐up models of expression‐fitness landscapes will require near‐exhaustive characterization of parts. SYNOPSIS Quantitative multiscale measurements of the responses to varied expression of translation termination factors in Bacillus subtilis reveal ‘regulatory entrenchment’, whereby expression‐fitness landscapes are dictated by idiosyncratic interactions in the regulatory network. Genetic tools and high‐throughput measurements are used to map translation termination factor (release factor) expression‐fitness landscapes in B. subtilis . Release factor depletion leads to imbalanced translation for co‐transcribed gene pairs. Imbalanced translation induces unintended regulons to the detriment of cell fitness. Swapping a single stop codon rewires global susceptibility to RF perturbation. The observed regulatory entrenchment presents a challenge for predictive bottom‐up models. Graphical Abstract Quantitative multiscale measurements of the responses to varied expression of translation termination factors in Bacillus subtilis reveal ‘regulatory entrenchment’, whereby expression‐fitness landscapes are dictated by idiosyncratic interactions in the regulatory network.