Characterization of interaction sites in the Saccharomyces cerevisiae ribosomal stalk components

Abstract

b and P2 a a a a 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-aminoacid-long C-end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1 a a a a with P2 b b b b and of P1 b b b b with P2 a a a a 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.

A Mode of Assembly of P0, P1, and P2 Proteins at the GTPase-associated Center in Animal Ribosome

“…After completion of this manuscript, Diaconu et al (36) reported the crystal structure of the L10⅐L7/L12 complex in Thermotoga martima, and the results surprisingly revealed three repetitive binding sites for L7/L12 dimers on the discrete C-terminal helix ␣-8. In yeast, the regions including residues 230 -290 (25), 212-262 (26), and 213-250 (27) within the C-terminal half of P0 have been identified as sites for P1/P2 binding (Fig. 8A), although individual sites for two P1-P2 dimers could not be resolved.…”

“…Current biochemical and genetic evidence indicates that P1 and P2 proteins form the heterodimer (16,(21)(22)(23)(24) and P1-P2 dimers bind to a specific region within the C-terminal domain of P0 (25)(26)(27). On the other hand, the rRNA binding site seems to be located within the N-terminal region comprising about 200 amino acids (25), although direct evidence has not been shown.…”

Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus

“…Multiple copies of the acidic ribosomal protein, or so-called stalk protein, are key components of this functional center (5-9). The stalk proteins form homo-or heterodimers, and two or three dimers bind to the ribosome through an anchor protein, L10 in bacteria and P0 in eukaryotes (7,(9)(10)(11)(12).In the case of bacteria, the structure and function of the stalk protein L7/L12 (termed L12 hereafter) is well established. The L12 protein is composed of an N-terminal dimerization domain and a globular C-terminal domain, which is connected by a flexible hinge region and has a wide range of movement (7, 13).…”

“…Multiple copies of the acidic ribosomal protein, or so-called stalk protein, are key components of this functional center (5-9). The stalk proteins form homo-or heterodimers, and two or three dimers bind to the ribosome through an anchor protein, L10 in bacteria and P0 in eukaryotes (7,(9)(10)(11)(12).…”

Ribosomes from Trypanosomatids: Unique Structural and Functional Properties

“…Functional complementation assays in yeast using C-terminal truncated P0 variants showed that deletion of 87 amino acids (the entire putative hydrophobic zipper) abolishes the binding of P1/P2 proteins to the ribosome [21]. Using yeast two-hybrid technique (Y2H), it has been shown that the 100 amino acid long C-terminal domain of P0 strongly interacts with P1 and P2 proteins [22]. However, the last 50 amino acids of P0 (including only two of the eight hydrophobic residues) were not able to interact with P1/P2.…”