title: Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli.
abstract: Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5΄-UTR sequences and introduced random and designed mutations into natural and artificially selected 5΄-UTRs. Several distinct properties could be ascribed to a group of 5΄-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5΄-UTRs.
authors.affiliation: Department of Chemistry, Faculty of Bioinformatics and Bioengeneering, Lomonosov Moscow State University, Moscow, 119992, Russia.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Evfratov
authors.affiliation: Department of Chemistry, Faculty of Bioinformatics and Bioengeneering, Lomonosov Moscow State University, Moscow, 119992, Russia.|Skolkovo Institute of Science and Technology, Skolkovo, Moscow, 143025, Russia.|A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.
authors.forename: Ilya A
authors.initials: IA
authors.lastname: Osterman
authors.affiliation: Department of Chemistry, Faculty of Bioinformatics and Bioengeneering, Lomonosov Moscow State University, Moscow, 119992, Russia.
authors.forename: Ekaterina S
authors.initials: ES
authors.lastname: Komarova
authors.affiliation: Department of Chemistry, Faculty of Bioinformatics and Bioengeneering, Lomonosov Moscow State University, Moscow, 119992, Russia.
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Pogorelskaya
authors.affiliation: Department of Chemistry, Faculty of Bioinformatics and Bioengeneering, Lomonosov Moscow State University, Moscow, 119992, Russia.|Skolkovo Institute of Science and Technology, Skolkovo, Moscow, 143025, Russia.|A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.
authors.forename: Maria P
authors.initials: MP
authors.lastname: Rubtsova
authors.affiliation: Department of Chemistry, Faculty of Bioinformatics and Bioengeneering, Lomonosov Moscow State University, Moscow, 119992, Russia.|Skolkovo Institute of Science and Technology, Skolkovo, Moscow, 143025, Russia.|A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.
authors.forename: Timofei S
authors.initials: TS
authors.lastname: Zatsepin
authors.affiliation: Research Institute for Physical-Chemical Medicine, FMBA, Moscow, 119435, Russia.
authors.forename: Tatiana A
authors.initials: TA
authors.lastname: Semashko
authors.affiliation: Research Institute for Physical-Chemical Medicine, FMBA, Moscow, 119435, Russia.|Moscow Institute of Physics and Technology, Dolgoprpudny, Moscow, 141700, Russia.
authors.forename: Elena S
authors.initials: ES
authors.lastname: Kostryukova
authors.affiliation: Department of Chemistry, Faculty of Bioinformatics and Bioengeneering, Lomonosov Moscow State University, Moscow, 119992, Russia.|A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Mironov
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo, Moscow, 143025, Russia.|A.A. Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow, 127051, Russia.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Burnaev
authors.affiliation: A.A. Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow, 127051, Russia.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Krymova
authors.affiliation: Department of Chemistry, Faculty of Bioinformatics and Bioengeneering, Lomonosov Moscow State University, Moscow, 119992, Russia.|Skolkovo Institute of Science and Technology, Skolkovo, Moscow, 143025, Russia.|A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.|A.A. Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow, 127051, Russia.|National Research University Higher School of Economics, Moscow, 123458, Russia.
authors.forename: Mikhail S
authors.initials: MS
authors.lastname: Gelfand
authors.affiliation: Research Institute for Physical-Chemical Medicine, FMBA, Moscow, 119435, Russia.
authors.forename: Vadim M
authors.initials: VM
authors.lastname: Govorun
authors.affiliation: Department of Chemistry, Faculty of Bioinformatics and Bioengeneering, Lomonosov Moscow State University, Moscow, 119992, Russia.|A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Bogdanov
authors.affiliation: Department of Chemistry, Faculty of Bioinformatics and Bioengeneering, Lomonosov Moscow State University, Moscow, 119992, Russia.|Skolkovo Institute of Science and Technology, Skolkovo, Moscow, 143025, Russia.|A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.
authors.forename: Petr V
authors.initials: PV
authors.lastname: Sergiev
authors.affiliation: Department of Chemistry, Faculty of Bioinformatics and Bioengeneering, Lomonosov Moscow State University, Moscow, 119992, Russia.|Skolkovo Institute of Science and Technology, Skolkovo, Moscow, 143025, Russia.|A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Dontsova
mesh_terms: D020121:5' Untranslated Regions; D002469:Cell Separation; D004926:Escherichia coli / Q000235:genetics*; D005434:Flow Cytometry; D017930:Genes, Reporter; D059014:High-Throughput Nucleotide Sequencing; D009154:Mutation; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000502:physiology; D014176:Protein Biosynthesis; D038621:Regulatory Sequences, Ribonucleic Acid
chemical_list: D020121:5' Untranslated Regions; D009711:Nucleotides; D038621:Regulatory Sequences, Ribonucleic Acid