STALL-seq: mRNA-display selection of bacterial and eukaryotic translational arrest sequences from large random-sequence libraries

The 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 Escherichia 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 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.