O'Loughlin et al. 2020 (PRJNA609751)

General Details

Title Polysomes bypass a 50 nucleotide coding gap less efficiently than monosomes due to attenuation of an unstable 5’ mRNA stem loop stimulator and enhanced drop-off
Organism
Number of Samples 2
Release Date 2020/03/02 00:00
Sequencing Types
Protocol Details

Study Links

Repository Details

SRA SRP251292
ENA SRP251292
GEO GSE146240
BioProject PRJNA609751

Publication

Title
Authors O'Loughlin S, Capece MC, Klimova M, Wills NM, Coakley A, Samatova E, O'Connor PBF, Loughran G, Weissman JS, Baranov PV, Rodnina MV, Puglisi JD, Atkins JF
Journal Journal of molecular biology
Publication Date 2020 Jul 24
Abstract Efficient translational bypassing of a 50-nt non-coding gap in a phage T4 topoisomerase subunit gene (gp60) requires several recoding signals. Here we investigate the function of the mRNA stem-loop 5' of the take-off codon, as well as the importance of ribosome loading density on the mRNA for efficient bypassing. We show that polysomes are less efficient at mediating bypassing than monosomes, both in vitro and in vivo, due to their preventing formation of a stem-loop 5' of the take-off codon and allowing greater peptidyl-tRNA drop off. A ribosome profiling analysis of phage T4-infected Escherichia coli yielded protected mRNA fragments within the normal size range derived from ribosomes stalled at the take-off codon. However, ribosomes at this position also yielded some 53-nucleotide fragments, 16 longer. These were due to protection of the nucleotides that form the 5' stem-loop. NMR shows that the 5' stem-loop is highly dynamic. The importance of different nucleotides in the 5' stem-loop is revealed by mutagenesis studies. These data highlight the significance of the 5' stem-loop for the 50-nt bypassing and further enhance appreciation of relevance of the extent of ribosome loading for recoding. Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.
PMC PMC7245268
PMID 32454154
DOI
Run Accession Study Accession Scientific Name Cell Line Library Type Treatment GWIPS-viz Trips-Viz Reads BAM BigWig (F) BigWig (R)
SRR11215525 PRJNA609751 Escherichia coli 0.0 Ribo-Seq 0.0
SRR11215528 PRJNA609751 Escherichia coli 0.0 Ribo-Seq 0.0
Run Accession Study Accession Scientific Name Cell Line Library Type Treatment GWIPS-viz Trips-Viz Reads BAM BigWig (F) BigWig (R)

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