Peer-Reviewed Journal Details
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O'Loughlin S.;Capece M.C.;Klimova M.;Wills N.M.;Coakley A.;Samatova E.;O'Connor P.B.F.;Loughran G.;Weissman J.S.;Baranov P.V.;Rodnina M.V.;Puglisi J.D.;Atkins J.F.
Journal of Molecular Biology
Polysomes Bypass a 50-Nucleotide Coding Gap Less Efficiently Than Monosomes Due to Attenuation of a 5' mRNA Stem豊oop and Enhanced Drop-off
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NMR spectroscopy recoding ribosome profiling structure translational bypassing
ゥ 2020 The Authors 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僕oop 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僕oop 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僕oop. NMR shows that the 5' stem僕oop is highly dynamic. The importance of different nucleotides in the 5' stem僕oop is revealed by mutagenesis studies. These data highlight the significance of the 5' stem僕oop for the 50-nt bypassing and further enhance appreciation of relevance of the extent of ribosome loading for recoding.
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