PT  - JOURNAL ARTICLE
AU  - Britta Seip
AU  - Guénaël Sacheau
AU  - Denis Dupuy
AU  - C Axel Innis
TI  - Ribosomal stalling landscapes revealed by high-throughput inverse toeprinting of mRNA libraries
AID  - 10.26508/lsa.201800148
DP  - 2018 Oct 01
TA  - Life Science Alliance
PG  - e201800148
VI  - 1
IP  - 5
4099  - https://www.life-science-alliance.org/content/1/5/e201800148.short
4100  - https://www.life-science-alliance.org/content/1/5/e201800148.full
SO  - Life Sci. Alliance2018 Oct 01; 1
AB  - Although it is known that the amino acid sequence of a nascent polypeptide can impact its rate of translation, dedicated tools to systematically investigate this process are lacking. Here, we present high-throughput inverse toeprinting, a method to identify peptide-encoding transcripts that induce ribosomal stalling in vitro. Unlike ribosome profiling, inverse toeprinting protects the entire coding region upstream of a stalled ribosome, making it possible to work with random or focused transcript libraries that efficiently sample the sequence space. We used inverse toeprinting to characterize the stalling landscapes of free and drug-bound Escherichia coli ribosomes, obtaining a comprehensive list of arrest motifs that were validated in vivo, along with a quantitative measure of their pause strength. Thanks to the modest sequencing depth and small amounts of material required, inverse toeprinting provides a highly scalable and versatile tool to study sequence-dependent translational processes.