title: Characterization of interaction sites in the Saccharomyces cerevisiae ribosomal stalk components.
abstract: The interactions among the yeast stalk components (P0, P1alpha, P1beta, P2alpha and P2beta) and with EF-2 have been explored using immunoprecipitation, affinity chromatography and the two-hybrid system. No stable association was detected between acidic proteins of the same type. In contrast, P1alpha and P1beta were found to interact with P2beta and P2alpha respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C-terminal P0 fragments. Immunoprecipitation shows the association of EF-2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two-hybrid assay. This interaction improves using the 100-amino-acid-long C-end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1alpha with P2beta and of P1beta with P2alpha rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid--CSIC, Cantoblanco, 28049 Madrid, Spain.
authors.forename: V S
authors.initials: VS
authors.lastname: Lalioti
authors.forename: J
authors.initials: J
authors.lastname: Pérez-Fernández
authors.forename: M
authors.initials: M
authors.lastname: Remacha
authors.forename: J P G
authors.initials: JP
authors.lastname: Ballesta
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002846:Chromatography, Affinity; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2 / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D020652:Peptide Elongation Factor 2; D010750:Phosphoproteins; D012269:Ribosomal Proteins; C031937:phosphoprotein P2, ribosomal; C035690:ribosomal phosphoprotein P1