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The ribosomal protein S1-dependent standby site in tisB mRNA consists of a single-stranded region and a 5 ' structure element
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.ORCID iD: 0000-0003-2771-0486
2019 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, no 32, p. 15901-15906Article in journal (Refereed) Published
Abstract [en]

In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 tisB mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In tisB and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient tisB mRNAs, we provide a thorough characterization of the tisB standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30 Delta S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional tisB standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 tisB mRNA (similar to 100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.

Place, publisher, year, edition, pages
NATL ACAD SCIENCES , 2019. Vol. 116, no 32, p. 15901-15906
Keywords [en]
translation initiation, ribosome standby site, RNA secondary structure, ribosomal protein S1, fluorescence anisotropy
National Category
Microbiology Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-392132DOI: 10.1073/pnas.1904309116ISI: 000478971900029PubMedID: 31320593OAI: oai:DiVA.org:uu-392132DiVA, id: diva2:1347979
Funder
Swedish Research CouncilEU, European Research CouncilKnut and Alice Wallenberg FoundationAvailable from: 2019-09-03 Created: 2019-09-03 Last updated: 2019-09-03Bibliographically approved

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