The Acidic Protein Binding Site Is Partially Hidden in the Free Saccharomyces cerevisiae Ribosomal Stalk Protein P0

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• 作者：Jorge Pé ; rez-Ferná ; ndez ; Miguel Remacha ; and Juan P. G. Ballesta
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：April 12, 2005
• 年：2005
• 卷：44
• 期：14
• 页码：5532 - 5540
• 全文大小：177K
• 年卷期：v.44,no.14(April 12, 2005)
• ISSN：1520-4995

文摘

The ribosomal stalk is essential for translation; however, its overall structure is poorlyunderstood. Characterization of the region involved in the interactions between protein P0 and the 12kDa acidic proteins P1 and P2 is fundamental to understand the assembly and function of this structurein the eukaryotic ribosome. The acidic protein content is important for the ribosome efficiency and affectsthe translation of specific mRNAs. By usage of a series of progressively truncated fragments of proteinP0 in the two-hybrid test, a region between positions 213 and 250 was identified as the minimal proteinpart able to interact with the acidic proteins. Extensions at either end affect the binding capacity of thefragment either positively or negatively depending on the number of added amino acids. Deletions insidethe binding region confirm its in vivo relevance since they drastically reduce the P0 interacting capacitywith the 12 kDa acidic proteins, which are severely reduced in the ribosome when the truncated proteinis expressed in the cell. Moreover, recombinant His-tagged P0 fragments containing the binding site andbound to Ni²⁺-NTA columns can form a complex with the P1 and P2 proteins, which is able to bindelongation factor EF2. The results indicate the existence of a region in P0 that specifically interacts withthe acidic proteins. These interactions are, however, hindered by the presence of neighbor protein domains,suggesting the need for conformational changes in the complete P0 to allow the assembly of the ribosomalstalk.