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dc.contributor.authorPekarek, Lukas
dc.contributor.authorBuck, Stefan
dc.contributor.authorCaliskan, Neva
dc.date.accessioned2022-04-11T12:06:41Z
dc.date.available2022-04-11T12:06:41Z
dc.date.issued2022-02-12
dc.date.submitted2021-03-23
dc.identifier.citationPekarek, L., Buck, S., Caliskan, N. Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation. J. Vis. Exp. (180), e62589, doi:10.3791/62589 (2022).en_US
dc.identifier.issn1940-087X
dc.identifier.urihttp://hdl.handle.net/10033/623171
dc.descriptionRNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously. This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.en_US
dc.description.abstractRNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously. This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.en_US
dc.description.sponsorshipThe work in our laboratory is supported by the Helmholtz Association and funding from the European Research Council (ERC) Grant Nr. 948636 (to NC). We thank Anuja Kibe and Jun. Prof. Redmond Smyth for critically reviewing the manuscript. We thank Tatyana Koch for expert technical assistance. We thank Kristyna Pekarkova for the help with recording experimental videos.en_US
dc.language.isoenen_US
dc.publisherJOVEen_US
dc.relationhttps://explore.openaire.eu/search/project?projectId=corda__h2020::55525a2777f46367fb0c6c0fa55b42f9en_US
dc.relation.urlhttps://dx.doi.org/10.3791/62589en_US
dc.rightsembargoedAccessen_US
dc.rightsAttribution-NonCommercial 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by-nc/4.0/*
dc.subjectmessenger RNA; conformation; genetics; human; optical tweezers; protein synthesisen_US
dc.subject.meshCOVID-19; Humans; Nucleic Acid Conformation; Optical Tweezers; Protein Biosynthesis; RNA, Messenger; SARS-CoV-2en_US
dc.titleOptical Tweezers to Study RNA-Protein Interactions in Translation Regulationen_US
dc.typeArticleen_US
dc.contributor.departmentHelmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Zentrum für Infektionsforschung (Helmholtz Centre for Infection Research), Germanyen_US
dc.identifier.journalJOVE: Journal of visualized Experimentsen_US


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