Direct and high throughput (HT) interactions on the ribosomal surface by iRIA.
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.
Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy.
Elisa
E
Pesce
Dept. of Immunology, Transplantation and Infectious Diseases, San Raffaele Research Institute, Milano, Italy.
Claudia
C
Minici
Biochemistry Center of Heidelberg University, Heidelberg, Germany.
Jochen
J
Baβler
Biochemistry Center of Heidelberg University, Heidelberg, Germany.
Ed
E
Hurt
Dept. of Immunology, Transplantation and Infectious Diseases, San Raffaele Research Institute, Milano, Italy.
Massimo
M
Degano
Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy.
Piera
P
Calamita
Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy.|Dept. of Biosciences, University of Milano, Milan, Italy.
Stefano
S
Biffo
D000818:Animals; D001156:Artemia / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D039642:Eukaryotic Initiation Factors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques / Q000379:methods*; D011485:Protein Binding / Q000235:genetics; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism
D039642:Eukaryotic Initiation Factors