Quantifying the performance and burden of genetic parts controlling translation using Ribo-seq

Ribosome binding sites and stop codons provide signals for where translation should start and stop, and secondary structures in messenger RNAs can stall ribosomes and induce frameshifting. We develop methods that use ribosome profiling to characterize parts controlling translation in situ. We simultaneously measure translation initiation rates and termination efficiencies across the Escherichia coli genome, in addition to the pausing and frameshifting of ribosomes at a strongly expressed synthetic RNA pseudoknot known to impart significant burden on the cell. We measure the transcriptional and translational response to this synthetic construct and find that sequestering of ribosomes at the pseudoknot leads to a σ32-mediated heat-shock response.