Direct and high throughput (HT) interactions on the ribosomal surface by iRIA

• Verfasst von: Pesce, Elisa [VerfasserIn]
• Verfasst von: Minici, Claudia [VerfasserIn]
• Verfasst von: Baßler, Jochen [VerfasserIn]
• Verfasst von: Hurt, Ed [VerfasserIn]
• Verfasst von: Degano, Massimo [VerfasserIn]
• Verfasst von: Calamita, Piera [VerfasserIn]
• Verfasst von: Biffo, Stefano [VerfasserIn]
• Titel: Direct and high throughput (HT) interactions on the ribosomal surface by iRIA
• Verf.angabe: Elisa Pesce, Claudia Minici, Jochen Baβler, Ed Hurt, Massimo Degano, Piera Calamita & Stefano Biffo
• E-Jahr: 2015
• Jahr: 21 October 2015
• Umfang: 8 S.
• Fussnoten: Gesehen am 02.07.2020
• Titel Quelle: Enthalten in: Scientific reports
• Ort Quelle: [London] : Macmillan Publishers Limited, part of Springer Nature, 2011
• Jahr Quelle: 2015
• Band/Heft Quelle: 5(2015) Artikel-Nummer 15401, 8 Seiten
• ISSN Quelle: 2045-2322
• DOI: doi:10.1038/srep15401
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1703266250

Abstract

Ribosomes function as platforms for binding of other molecules, but technologies for studying this process are lacking. Therefore we developed iRIA (in vitro Ribosomes Interaction Assay). In approach I, Artemia salina ribosomes spotted on solid phase are used for binding picomoles of analytes; in approach II, cellular extracts allow the measurement of ribosome activity in different conditions. We apply the method to analyze several features of eIF6 binding to 60S subunits. By approach I, we show that the off-rate of eIF6 from preribosomes is slower than from mature ribosomes and that its binding to mature 60S occurs in the nM affinity range. By approach II we show that eIF6 binding sites on 60S are increased with mild eIF6 depletion and decreased in cells that are devoid of SBDS, a ribosomal factor necessary for 60S maturation and involved in Swachman Diamond syndrome. We show binding conditions to immobilized ribosomes adaptable to HT and quantify free ribosomes in cell extracts. In conclusion, we suggest that iRIA will greatly facilitate the study of interactions on the ribosomal surface.