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  Polysomes bypass a 50-nucleotide coding gap less efficiently than monosomes due to attenuation of a 5′ mRNA stem–loop and enhanced drop-off

O'Loughlin, S., Capece, M. C., Klimova, M., Wills, N. M., Coakley, A., Samatova, E. N., et al. (2020). Polysomes bypass a 50-nucleotide coding gap less efficiently than monosomes due to attenuation of a 5′ mRNA stem–loop and enhanced drop-off. Journal of Molecular Biology, 432(16), 4369-4387. doi:10.1016/j.jmb.2020.05.010.

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Item Permalink: http://hdl.handle.net/21.11116/0000-0007-2C26-E Version Permalink: http://hdl.handle.net/21.11116/0000-0007-2C2F-5
Genre: Journal Article

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 Creators:
O'Loughlin, S., Author
Capece, M. C., Author
Klimova, M.1, Author              
Wills, N. M., Author
Coakley, A., Author
Samatova, E. N.1, Author              
O'Connor, P.k B. F., Author
Loughran, G., Author
Weissman, J. S., Author
Baranov, P. V., Author
Rodnina, M. V.2, Author              
Puglisi, J.h D., Author
Atkins, J. F., Author
Affiliations:
1Department of Physical Biochemistry, MPI for Biophysical Chemistry, Max Planck Society, ou_578598              
2Department of Physical Biochemistry, MPI for biophysical chemistry, Max Planck Society, ou_578598              

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Free keywords: NMR spectroscopy; structure; ribosome profiling; translational bypassing; recoding
 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.

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Language(s): eng - English
 Dates: 2020-05-232020-07-24
 Publication Status: Published in print
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 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.jmb.2020.05.010
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Title: Journal of Molecular Biology
Source Genre: Journal
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Pages: - Volume / Issue: 432 (16) Sequence Number: - Start / End Page: 4369 - 4387 Identifier: -