Translational arrest is a phenomenon wherein a temporary pause or slowing of the translation elongation reaction occurs due to the interaction between ribosome and nascent peptide. Recent studies have revealed that translational arrest peptides are involved in intracellular protein homeostasis regulatory functions, such as gene expression regulation at the translational level and regulation of cotranslational protein folding. Herein, we established a method for the large-scale in vitro selection of translational arrest peptides from DNA libraries by combining a modified mRNA display method and deep sequencing. We performed in vitro selection of translational arrest sequences from random-sequence libraries via mRNA display based on the E. coli PURE system or wheat germ extract. Following several rounds of affinity selection, we obtained various candidate sequences that were not similar to known arrest peptides and subsequently confirmed their ribosome stalling activity by peptidyl-tRNA detection and toeprinting assay. Following the site-directed mutagenesis of the selected sequences, these clones were found to contain novel arrest peptide motifs. This method, termed as STALL-seq (Selection of Translational Arrest sequences from Large Library sequencing), could be useful for the large-scale investigation of translational arrest sequences acting on both bacterial and eukaryotic ribosomes and could help discover novel intracellular regulatory mechanisms.

中文翻译：

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STALL-seq：从大型随机序列库中选择细菌和真核生物翻译阻滞序列的 mRNA 展示。


翻译停滞是由于核糖体和新生肽之间的相互作用而发生翻译伸长反应暂时暂停或减慢的现象。最近的研究表明，翻译期阻滞肽参与细胞内蛋白质稳态调节功能，例如翻译水平的基因表达调控和共翻译蛋白折叠的调节。在此，我们建立了一种通过结合修饰的 mRNA 展示方法和深度测序从 DNA 文库中大规模体外选择翻译阻滞肽的方法。我们通过基于大肠杆菌 PURE 系统或小麦胚芽提取物的 mRNA 展示从随机序列文库中对翻译停滞序列进行了体外选择。经过几轮亲和选择，我们获得了与已知阻滞肽不相似的各种候选序列，随后通过肽基-tRNA 检测和脚趾印测定证实了它们的核糖体停滞活性。在对所选序列进行定点诱变后，发现这些克隆包含新的阻滞肽基序。这种方法被称为 STALL-seq（从大文库测序中选择翻译阻滞序列），可用于大规模研究作用于细菌和真核核糖体的翻译阻滞序列，并可能有助于发现新的细胞内调节机制。