Flow-Seq Evaluation of Translation Driven by a Set of Natural Escherichia coli 5′-UTR of Variable Length

Flow-seq is a method that combines fluorescently activated cell sorting and next-generation sequencing to deduce a large amount of data about translation efficiency from a single experiment. Here, we constructed a library of fluorescent protein-based reporters preceded by a set of 648 natural 5 '-untranslated regions (5 '-UTRs) of Escherichia coli genes. Usually, Flow-seq libraries are constructed using uniform-length sequence elements, in contrast to natural situations, where functional elements are of heterogenous lengths. Here, we demonstrated that a 5 '-UTR library of variable length could be created and analyzed with Flow-seq. In line with previous Flow-seq experiments with randomized 5 '-UTRs, we observed the influence of an RNA secondary structure and Shine-Dalgarno sequences on translation efficiency; however, the variability of these parameters for natural 5 '-UTRs in our library was smaller in comparison with randomized libraries. In line with this, we only observed a 30-fold difference in translation efficiency between the best and worst bins sorted with this factor. The results correlated with those obtained with ribosome profiling.

Automated summary

Flow-seq is a method that combines fluorescently activated cell sorting and next-generation sequencing to deduce a large amount of data about translation efficiency from a single experiment. It has been demonstrated that a variable length 5'-UTR library can be created and analyzed with Flow-seq, and the influence of RNA secondary structure and Shine-Dalgarno sequences on translation efficiency is smaller for natural 5'-UTRs compared to randomized libraries.