Spurious regulatory connections dictate the expression-fitness landscape of translation termination factors

Abstract

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 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.HighlightsPrecision measurements enable multiscale expression-to-fitness mapping.RF 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.

Translational activation by an alternative sigma factor inBacillus subtilis

“…For matched Rend-seq/ribosome profiling datasets, strains were grown in LB or conditioned MCC medium (Parker et al 2020; Lalanne et al 2020) with various inducer (xylose, IPTG) concentrations (see Table S1). For these datasets, cells were grown in exponential phase for at least 10 doublings before harvesting at OD 600 ≈0.3.…”

“…Matched Rend-seq and ribosome profiling datasets used to identify genes with increased TE (Figure 1) and to estimate the short isoform fraction and corresponding apparent TE (Figure 3, Figure S3) were obtained from GEO accession GSE162169 (Lalanne et al 2020). These datasets display a range of σ B activation due to a diverse set of genetic modifications and growth media.…”

“…These datasets display a range of σ B activation due to a diverse set of genetic modifications and growth media. In particular, we previously identified that tuning the expression of translation termination factors RF2 and PrmC activate σ B to varying degrees (Lalanne et al 2020). For example, the σ B active data presented in Figures 1 and 2 correspond to a CRISPRi knockdown of RF2, while σ B inactive corresponds to wild-type.…”

“…For example, the σ B active data presented in Figures 1 and 2 correspond to a CRISPRi knockdown of RF2, while σ B inactive corresponds to wild-type. Importantly, although it is possible that different RF2 levels could affect translation initiation (and therefore TE) of genes (Lalanne et al 2020), none of the genes that show a substantial increase in TE (Figure 1) have a UGA stop codon or are co-transcribed with a gene ending with UGA stop (UGA being the stop codon cognate to RF2). Hence, the molecular causes of σ B activation are distinct and independent from the mechanisms leading to translational activation characterized here.…”

First-principles model of optimal translation factors stoichiometry

“…As a side note, the expression for termination time τ in equation 6 must be modified in a regime where ribosomes are frequently queued upstream of stop codons. This would occur if the termination rate were slow and approached initiation rates on mRNAs [7,33]. In this regime, queues of ribosomes at stop codons would incur an additional time to terminate.…”