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

Abstract

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.

Flow-Seq Method: Features and Application in Bacterial Translation Studies

“…Similar observations relating to the low stability of the secondary structure and the conservation of the SD sequence were made for the variants determining a high translation efficiency [ 2 ]. The same factors were found to be significant for the translation efficiency of the reporter gene preceded by a set of natural 5’ UTRs; however, in this case, the variability of the translation efficiency was much lower than it was for the library of fully randomized 5’ UTR sequences [ 77 ]. There were also individual cases being indicative of the presence of AU-rich enhancers at the 5’ terminus at the standby site, low abundance of cytidine bases, multiple SD sequences, and AG repeats in the mRNA 5’ UTRs, which provide the high reporter protein level [ 2 ].…”

“…The Flow-seq method has been repeatedly used to gain insight into how the nucleotide sequences of different motifs of 5’ UTRs affect the translation efficiency. In particular, the ribosome binding sites with a fixed SD sequence [ 74 ], 5’ UTRs of different fixed lengths [ 2 ], or natural 5’ UTRs of different lengths [ 77 ], as well as standby sites and spacer regions [ 72 ], were studied. An analysis of tens of thousands of tested variants showed that the variation in the efficiency of the reporter protein synthesis can amount to four, and even five, orders of magnitude.…”

“…Flow-seq многократно использовался для того, чтобы понять, как нуклеотидные последовательности различных участков 5'-UTR влияют на эффективность трансляции. В частности, такому исследованию подвергали сайты посадки рибосомы с фиксированной последовательностью SD [74], 5'-UTR разной фиксированной длины [2] или природные 5'-UTR разной длины [77], а также standbyсайты и спейсерные участки [72]. Анализ десятков тысяч проверенных вариантов позволил получить разницу в пределах 4 и даже 5 порядков в эффективности синтеза репортерного белка.…”

“…Схожие наблюдения, связанные с низкой стабильностью вторичной структуры и консервативностью последовательности SD, прослеживались в случае вариантов, определяющих высокую эффективность трансляции [2]. Те же факторы выявлены как значимые для эффективности трансляции репортерного гена, перед которым находился набор природных 5'-UTR, однако в данном случае вариабельность эффективности трансляции была значительно меньше, чем в случае библиотеки полностью рандомизованных последовательностей 5'-UTR [77]. Встречались и отдельные случаи, свидетельствовавшие о присутствии AU-богатых энхансеров на 5'-конце в standby-сайте, низкой доле цитидиновых оснований, множественных SD, AGповторов в 5'-UTR мРНК, обеспечивающих высокий уровень репортерного белка [2].…”

Influence of 5’-UTR base types on translation efficiency in Escherichia coli

“…Multiple high-throughput assays have been developed to validate the effect of 5'-UTR on protein translation rate, such as the Flow-seq and ribosome profiling (Ingolia et al 2009;Komarova et al 2022). Previous studies have concentrated on constructing 5'-UTR random libraries to screen out artificial 5'-UTR with high translation efficiency or exploring the influences of 5'-UTR sequence characteristics on protein translation efficiency by Flow-seq, such as secondary structure, the hybridization of the 16S rRNA to the RBS, AU enhancer (Osterman et al 2013).…”