Dataset Information

Ribosomal stalling landscapes revealed by high-throughput inverse toeprinting of mRNA libraries.

ABSTRACT: 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.

SUBMITTER: Seip B

PROVIDER: S-EPMC6238534 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Ribosomal stalling landscapes revealed by high-throughput inverse toeprinting of mRNA libraries.

Seip Britta B Sacheau Guénaël G Dupuy Denis D Innis C Axel CA

Life science alliance 20181009 5

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 transcrip ...[more]

PMID: 30456383

Similar Datasets

Project description:The huge chemical space potential of synthetic polymers means that in many studies only a small parameter range can be realistically synthesized in a short time and hence the "best" formulations may not be optimum. Throughput is traditionally limited by the need for deoxygenation in radical polymerizations, but advances in photopolymerization now provide opportunities for "in-air" polymerizations. Here, we have developed a protocol using liquid handling robots (or multichannel pipettes) with blue light photolysis of reversible addition fragmentation chain transfer agents and tertiary amine deoxygenation to enable the synthesis of polymer libraries in industry-standard 96-well plates with no specialized infrastructure and no degassing step. The roles of solvents and amine deoxygenators are explored to optimize the polymerization, particularly to look at alternatives to dimethyl sulfoxide (DMSO) for hydrophobic monomer (co)polymerization. DMSO is shown to aid the degassing process but is not easy to remove and hence prevents isolation of pure polymers. In contrast, using dioxane in-plate evaporation or precipitation of the tertiary amine allowed isolation of polymers in-plate. This removed all reaction components yielding pure polymers, which is not easily achieved with systems using metal catalysts and secondary reductants, such as ascorbic acid. As an example of the throughput, in just under 40 h, 392 polymers were synthesized and subsequently analyzed direct from plates by a 96-well plate sampling size exclusion chromatography system to demonstrate reproducibility. Due to less efficient degassing in dioxane (compared to DMSO), the molecular weight and dispersity control were limited in some cases (with acrylates giving the lowest dispersities), but the key aim of this system is to access hundreds of polymers quickly and in a format needed to enable testing. This method enables easy exploration of chemical space and development of screening libraries to identify hits for further study using precision polymerization methods.

Dataset Information

Ribosomal stalling landscapes revealed by high-throughput inverse toeprinting of mRNA libraries.

Publications

Ribosomal stalling landscapes revealed by high-throughput inverse toeprinting of mRNA libraries.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets