Characterizing ribosomal stalling landscapes in vitro

Seip, B., Sacheau, G., Dupuy, D., Innis, C.A. (2018). Ribosomal stalling landscapes revealed by high-throughput inverse toeprinting of mRNA libraries. Life Science Alliance 1 (5).

We present inverse toeprinting (now referred to as iTP-Seq), an in vitro profiling method that locates stalled ribosomes on the mRNA with codon-level resolution. This technique is based on the use of a highly processive 3’ to 5’ RNA exonuclease (RNase R), such that the leading ribosome stalled on a given transcript protects the mRNA upstream of the arrest site from degradation. Since the entire peptide-encoding sequence is preserved, prior knowledge of the transcript sequences is not required. Thus, the nature and complexity of the input mRNA library can be tuned for specific purposes, such as the large-scale characterization of context-dependent antibiotics or the deep mutational scanning of specific translation arrest-inducing sequences.