A predictive biophysical model of translationalcoupling to coordinate and control protein expressionin bacterial operons

Natural and engineered genetic systems requirethe coordinated expression of proteins. In bacteria, translational coupling provides a genetically encodedmechanism to control expression level ratioswithin multi-cistronic operons. We have developeda sequence-to-function biophysical model of translationalcoupling to predict expression level ratiosin natural operons and to design synthetic operonswith desired expression level ratios. To quantitativelymeasure ribosome re-initiation rates, wedesigned and characterized 22 bi-cistronic operonvariants with systematically modified intergenic distancesand upstream translation rates. We then deriveda thermodynamic free energy model to calculatede novo initiation rates as a result of ribosomeassistedunfolding of intergenic RNA structures. Thecomplete biophysical model has only five free parameters, but was able to accurately predict downstreamtranslation rates for 120 synthetic bi-cistronicand tri-cistronic operons with rationally designed intergenicregions and systematically increased upstreamtranslation rates. The biophysical model alsoaccurately predicted the translation rates of the nineprotein atp operon, compared to ribosome profilingmeasurements. Altogether, the biophysical modelquantitatively predicts how translational couplingcontrols protein expression levels in synthetic andnatural bacterial operons, providing a deeper understandingof an important post-transcriptional regulatorymechanism and of fering the ability to rationallyengineer operons with desired behaviors.