Characterization of the 26S rRNA-binding domain in Saccharomyces cerevisiae ribosomal stalk phosphoprotein P0.
The stalk is a universal structure of the large ribosomal subunit involved in the function of translation factors. The bacterial stalk is highly stable but its stability is notably reduced in eukaryotes, favouring a translation regulatory activity of this ribosomal domain, which has not been reported in prokaryotes. The RNA-binding protein P0 plays a key role in determining the eukaryotic stalk activities, and characterization of the P0/RNA interaction is essential to understand the evolutionary process. Using a series of Saccharomyces cerevisiae-truncated proteins, a direct involvement of two N-terminal regions, I3-M58 and K81-V121, in the interaction of P0 with the ribosome has been shown. Two other conserved regions, R122-T149 and G162-T182, affect P0 interaction with other stalk components and the sensitivity to sordarin anti-fungals but are not essential for RNA binding. Moreover, P0 and a P0 fragment comprising only the first 121 amino acids show a similar in vitro affinity for the highly conserved 26S rRNA binding site. A protein chimera containing the first 165 amino acids of L10, the P0 bacterial counterpart, is able to complement the absence of P0 and also shows the same P0 RNA binding characteristics. Altogether, the results indicate that the affinity of the stalk RNA-binding protein for its substrate has been highly conserved, and changes in the stability of the interaction of P0 with the ribosome, which are essential for the new eukaryotic functions, result from the evolution of the overall stalk structure.
Santos
Cruz
C
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco 28049, Madrid, Spain.
Ballesta
Juan P G
JP
D000935:Antifungal Agents / Q000494:pharmacology; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D007192:Indenes / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D012335:RNA, Ribosomal / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D000935:Antifungal Agents; D005656:Fungal Proteins; D007192:Indenes; D010750:Phosphoproteins; D012335:RNA, Ribosomal; C065096:RNA, ribosomal, 26S; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C054675:ribosomal protein P0; C003329:sordarin