Polysomes Bypass a 50-Nucleotide Coding Gap Less Efficiently Than Monosomes Due to Attenuation of a 5′ mRNA Stem–Loop and Enhanced Drop-off. Issue 16 (24th July 2020)

• Record Type: Journal Article
• Authors: O'Loughlin, Sinéad; Capece, Mark C.; Klimova, Mariia; Wills, Norma M.; Coakley, Arthur; Samatova, Ekaterina; O'Connor, Patrick B.F.; Loughran, Gary; Weissman, Jonathan S.; Baranov, Pavel V.; Rodnina, Marina V.; Puglisi, Joseph D.; Atkins, John F.
• Is Part Of: Journal of molecular biology. Volume 432:Issue 16(2020)
• Page Start: 4369
• Page End: 4387
• Publication Date: 2020-07-24
• Subjects: NMR spectroscopy -- structure -- ribosome profiling -- translational bypassing -- recoding; ORF open reading frame -- smFRET single-molecule fluorescence resonance energy transfer -- cryo-EM cryogenic electron microscopy -- SD Shine–Dalgarno -- SHAPE Selective 2′-Hydroxyl Acylation analyzed by Primer Extension -- RNase ribonuclease -- NOESY nuclear Overhauser enhancement spectroscopy -- COSY correlated spectroscopy; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2020.05.010
• Languages: English
• ISSNs: 0022-2836

Abstract

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. Graphical abstract: Unlabelled Image Highlights: Monosomes are more efficient than polysome in mediating 50-nt translational bypassing. A 5′ mRNA stem–loop facilitates translational bypassing by monosomes. Ribosome profiling yields an extra-long, 53-nt, protected fragment of mRNA.