A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons

Abstract

Natural and engineered genetic systems require the coordinated expression of proteins. In bacteria, translational coupling provides a genetically encoded mechanism to control expression level ratios within multi-cistronic operons. We have developed a sequence-to-function biophysical model of translational coupling to predict expression level ratios in natural operons and to design synthetic operons with desired expression level ratios. To quantitatively measure ribosome re-initiation rates, we designed and characterized 22 bi-cistronic operon variants with systematically modified intergenic distances and upstream translation rates. We then derived a thermodynamic free energy model to calculate de novo initiation rates as a result of ribosome-assisted unfolding of intergenic RNA structures. The complete biophysical model has only five free parameters, but was able to accurately predict downstream translation rates for 120 synthetic bi-cistronic and tri-cistronic operons with rationally designed intergenic regions and systematically increased upstream translation rates. The biophysical model also accurately predicted the translation rates of the nine protein atp operon, compared to ribosome profiling measurements. Altogether, the biophysical model quantitatively predicts how translational coupling controls protein expression levels in synthetic and natural bacterial operons, providing a deeper understanding of an important post-transcriptional regulatory mechanism and offering the ability to rationally engineer operons with desired behaviors.

Engineering Escherichia coli for propionic acid production through the Wood–Werkman cycle

“…For multimeric enzymes (mutase and carboxyl transferase), the values were adjusted to follow the stoichiometry for each subunit. The functionality of the operon was evaluated using the online operon calculator tool (https://salislab.net/software/predict_operon_calculator; Espah Borujeni et al, ; Tian & Salis, ). T7 promoter constrained the use of pMA_WWCV1 to BL21.…”

“…The mutase consists of two subunits in a 1:1 ratio (Mancia, Smith, & Evans, 1999), while the transcarboxylase requires 30 subunits at different ratios (Carey, Sönnichsen, & Yee, 2004). In the initial two-operon system (WWCV1), the RBSs were designed individually to provide similar translation rates (Espah Borujeni et al, 2013;Salis et al, 2009;Tian & Salis, 2015). However, using the reverse engineering RBS calculator tool, the operon showed considerable differences in the translation rates.…”

“…Each synthetic operon presents the following structure: A T7 promoter, a modified anaerobic nar promoter (Walker & DeMoss, 1992), a strong ribosomal-binding site (RBS) derived from the −10 consensus region of the nar promoter, the corresponding coding sequence, an additional stop sequence TAA, and a T7 terminator. The design of RBS and the analysis of operon translation rate were performed using the online tool RBS calculator (Espah Borujeni, Channarasappa, & Salis, 2013;Salis, Mirsky, & Voigt, 2009;Tian & Salis, 2015). RBS tuning down was performed considering all the genes in a single messenger RNA.…”

Toward a genetic tool development pipeline for host-associated bacteria

“…One promising solution is the use of in silico biophysical models to predict expression strengths based on sequence information (Figure 3B). The Salis lab has developed an online suite of software (https://salislab.net/software/) to predict protein expression levels based on the energetics of mRNA folding, ribosome-mRNA binding, and translational initiation as well as the impact of standby sites and translational coupling [30–33]. Aside from predicting translation strengths, these algorithms can design small libraries of RBS sequences covering a broad expression range to reduce the number of variants to be screened for pathway optimization.…”

Eukaryotic and prokaryotic gene structure

“…Some operons also display translational coupling, where the translation rates of multiple ORFs within an operon are linked. [28] This can occur when the ribosome remains attached at the end of an ORF and simply translocates along to the next without the need for a new RBS. [29] Translational coupling is also observed when translation of an ORF affects the accessibility of the next RBS through changes in RNA secondary structure.…”