Polysomes Bypass a 50-Nucleotide Coding Gap Less Efficiently Than Monosomes Due to Attenuation of a 5' mRNA Stem-Loop and Enhanced Drop-off
Autor: | Sinéad O’Loughlin, Ekaterina Samatova, Patrick B F O'Connor, Pavel V. Baranov, Jonathan S. Weissman, Arthur Coakley, John F. Atkins, Gary Loughran, Joseph D. Puglisi, Marina V. Rodnina, Mariia Klimova, Norma M. Wills, Mark C. Capece |
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Rok vydání: | 2020 |
Předmět: |
Models
Molecular medicine.disease_cause Ribosome profiling Ribosome Article 03 medical and health sciences Viral Proteins 0302 clinical medicine NMR spectroscopy Structural Biology Polysome medicine Escherichia coli Bacteriophage T4 Nucleotide RNA Messenger Molecular Biology Gene 60 030304 developmental biology chemistry.chemical_classification 0303 health sciences Messenger RNA Chemistry Recoding Structure Stem-loop Magnetic Resonance Imaging In vitro Cell biology RNA Bacterial Translational bypassing Polyribosomes Nucleic Acid Conformation 030217 neurology & neurosurgery |
Zdroj: | Journal of Molecular Biology |
ISSN: | 1089-8638 |
Popis: | 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 E. 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 |
Databáze: | OpenAIRE |
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