Место издания:Общество с ограниченной ответственностью "Академиздат"
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Аннотация:Protein biosynthesis by the ribosomes is not only an important step of gene expression andits regulation, but also a target for multiple antibiotics. Monitoring of translation efficiency andribosome progression along mRNAs might by addressed by several methods based on nextgeneration sequencing. The most known is an in vivo ribosome profiling. However, latter methodsuffers from several limitations, such as too wide range of different mRNA abundancies and inabilityto address an influence of a particular mRNA feature while keeping other properties identical.For a long time, a reverse transcription-based toe-printing has been a standard low throughputmethod to monitor ribosome movement along a particular model mRNA. Ribosome profiling andtoe-printing allowed several laboratories to reveal that a number of antibiotics acting upon theribosome inhibit translation in a sequence-specific manner, which was absolutely unanticipatedpreviously [1, 2]. Such studies remained sparse due to the limitations of both methods. Toinvestigate this issue comprehensively, we have developed a new method called Toe-Seq.Toe-Seq make use of barcoded mRNA libraries with randomized regions. Reversetranscription of mRNAs being translated in vitro allowed us to unequivocally identify mRNAdue to a barcode in its 3’-untranslated region and position ribosome by the length of cDNAproduct. This method has been applied to ribosome stalling by a variety of the followingantibiotics: tetracycline, hygromycin B, spectinomycin and amicoumacin A targeting the smallribosomal subunit, and erythromycin, chloramphenicol, tetracenomycin X and etamycin Abinding to the 50S ribosomal subunit. Intriguingly, for most of them, a distinctive contextspecificity can be observed. For chloramphenicol and erythromycin our data are consistentwith those obtained earlier using the ribosome profiling technique [2, 3], thus validating themethod of Toe-Seq. For a number of other antibiotics, the data on a combination of sequenceand positional specificity are completely new. The Toe-Seq method allowed us to additionallyassess other properties of mRNAs, such as efficiency of translation initiation and re-initiationas well as to identify natural antibiotic-independent ribosome pausing sites.Toe-Seq method proved to be a useful tool which might have a profound influence on theresearch of protein biosynthesis.
