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

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′-unt...

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Bibliographic Details
Published in:International journal of molecular sciences Vol. 23; no. 20; p. 12293
Main Authors: Komarova, Ekaterina S., Slesarchuk, Anna N., Rubtsova, Maria P., Osterman, Ilya A., Tupikin, Alexey E., Pyshnyi, Dmitry V., Dontsova, Olga A., Kabilov, Marsel R., Sergiev, Petr V.
Format: Journal Article
Language:English
Published: Basel MDPI AG 14-10-2022
MDPI
Subjects:
Binding sites
Cloning
E coli
Efficiency
Escherichia coli
Flow-seq
Fluorescence
Gene expression
Libraries
Next-generation sequencing
Protein structure
Proteins
ribosome
Secondary structure
Translation
translation efficiency
untranslated region
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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. 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.
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content type line 23
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms232012293