Absolute quantification of translational regulation and burden using combined sequencing approaches

<jats:title>Abstract</jats:title><jats:p>Translation of mRNAs into proteins is a key cellular process. Ribosome binding sites and stop codons provide signals to initiate and terminate translation, while stable secondary mRNA structures can induce translational recoding events. Fluorescent proteins are commonly used to characterize such elements but require the modification of a part’s natural context and allow only a few parameters to be monitored concurrently. Here, we develop an approach that combines ribosome profiling (Ribo-seq) with quantitative RNA sequencing (RNA-seq) to enable the high-throughput characterization of genetic parts controlling translation in absolute units. We simultaneously measure 743 translation initiation rates and 746 termination efficiencies across the <jats:italic>Escherichia coli</jats:italic> transcriptome, in addition to translational frameshifting induced at a stable RNA pseudoknot structure. By analyzing the transcriptional and translational response, we discover that sequestered ribosomes at the pseudoknot contribute to a σ<jats:sup>32</jats:sup>-mediated stress response, codon-specific pausing, and a drop in translation initiation rates across the cell. Our work demonstrates the power of integrating global approaches towards a comprehensive and quantitative understanding of gene regulation and burden in living cells.</jats:p>