Structural characterization of the ribosomal P1A-P2B protein dimer by small-angle X-ray scattering and NMR spectroscopy

The five ribosomal P-proteins, denoted P0-(P1-P2)2, constitute the stalk structure of the large subunit of eukaryotic ribosomes. In the yeast Saccharomyces cerevisiae, the group of P1 and P2 proteins is differentiated into subgroups that form two separate P1A-P2B and P1B-P2A heterodimers on the stalk. So far, structural studies on the P-proteins have not yielded any satisfactory information using either X-ray crystallography or NMR spectroscopy, and the structures of the ribosomal stalk and its individual constituents remain obscure. Here we outline a first, coarse-grained view of the P1A-P2B solution structure obtained by a combination of small-angle X-ray scattering and heteronuclear NMR spectroscopy. The complex has an elongated shape with a length of 10 nm and a cross section of approximately 2.5 nm. 15N NMR relaxation measurements establish that roughly 30% of the residues are present in highly flexible segments, which belong primarily to the linker region and the C-terminal part of the polypeptide chain. Secondary structure predictions and NMR chemical shift analysis, together with previous results from CD spectroscopy, indicate that the structured regions involve alpha-helices. NMR relaxation data further suggest that several helices are arranged in a nearly parallel or antiparallel topology. These results provide the first structural comparison between eukaryotic P1 and P2 proteins and the prokaryotic L12 counterpart, revealing considerable differences in their overall shapes, despite similar functional roles and similar oligomeric arrangements. These results present for the first time a view of the structure of the eukaryotic stalk constituents, which is the only domain of the eukaryotic ribosome that has escaped successful structural characterization.     

Assembly of Saccharomyces cerevisiae ribosomal stalk: binding of P1 proteins is required for the interaction of P2 proteins

The yeast ribosomal stalk is formed by a protein pentamer made of the 38 kDa P0 and four 12 kDa acidic P1/P2. The interaction of recombinant acidic proteins P1 alpha and P2 beta with ribosomes from Saccharomyces cerevisiae D4567, lacking all the 12 kDa stalk components, has been used to study the in vitro assembly of this important ribosomal structure. Stimulation of the ribosome activity was obtained by incubating simultaneously the particles with both proteins, which were nonphosphorylated initially and remained unmodified afterward. The N-terminus state, free or blocked, did not affect either the binding or reactivating activity of both proteins. Independent incubation with each protein did not affect the activity of the particles, however, protein P2 beta alone was unable to bind the ribosome whereas P1 alpha could. The binding of P1 alpha alone is a saturable process in acidic-protein-deficient ribosomes and does not take place in complete wild-type particles. Binding of P1 proteins in the absence of P2 proteins takes also place in vivo, when protein P1 beta is overexpressed in S. cerevisiae. In contrast, protein P2 beta is not detected in the ribosome in the P1-deficient D67 strain despite being accumulated in the cytoplasm. The results confirm that neither phosphorylation nor N-terminal blocking of the 12 kDa acidic proteins is required for the assembly and function of the yeast stalk. More importantly, and regardless of the involvement of other elements, they indicate that stalk assembling is a coordinated process, in which P1 proteins would provide a ribosomal anchorage to P2 proteins, and P2 components would confer functionality to the complex.     

P1 and P2 protein heterodimer binding to the P0 protein of Saccharomyces cerevisiae is relatively non-specific and a source of ribosomal heterogeneity

The ribosomal stalk is formed by four acidic phosphoproteins in Saccharomyces cerevisiae, P1α, P1β, P2α and P2β, which form two heterodimers, P1α/P2β and P1β/P2α, that preferentially bind to sites A and B of the P0 protein, respectively. Using mutant strains carrying only one of the four possible P1/P2 combinations, we found a specific phenotype associated to each P1/P2 pair, indicating that not all acidic P proteins play the same role. The absence of one P1/P2 heterodimer reduced the rate of cell growth by varying degrees, depending on the proteins missing. Synthesis of the 60S ribosomal subunit also decreased, particularly in strains carrying the unusual P1α-P2α or P1β-P2β heterodimers, although the distinct P1/P2 dimers are bound with similar affinity to the mutant ribosome. While in wild-type strains the B site bound P1β/P2α in a highly specific manner and the A site bound the four P proteins similarly, both the A and B binding sites efficiently bound practically any P1/P2 pair in mutant strains expressing truncated P0 proteins. The reported results support that while most ribosomes contain a P1α/P2β-P0-P1β/P2α structure in normal conditions, the stalk assembly mechanism can generate alternative compositions, which have been previously detected in the cell.     

Analysis of the protein-protein interactions between the human acidic ribosomal P-proteins: evaluation by the two hybrid system

The surface acidic ribosomal proteins (P-proteins), together with ribosomal core protein P0 form a multimeric lateral protuberance on the 60 S ribosomal subunit. This structure, also called stalk, is important for efficient translational activity of the ribosome. In order to shed more light on the function of these proteins, we are the first to have precisely analyzed mutual interactions among human P-proteins, employing the two hybrid system. The human acidic ribosomal P-proteins, (P1 or P2,) were fused to the GAL4 binding domain (BD) as well as the activation domain (AD), and analyzed in yeast cells. It is concluded that the heterodimeric complex of the P1/P2 proteins is formed preferentially. Formation of homodimers (P1/P1 and P2/P2) can also be observed, though with much less efficiency. Regarding that, we propose to describe the double heterodimeric complex as a protein configuration which forms the 60 S ribosomal stalk: P0-(P1/P2)(2). Additionally, mutual interactions among human and yeast P-proteins were analyzed. Heterodimer formation could be observed between human P2 and yeast P1 proteins.     

Yeast ribosomal P0 protein has two separate binding sites for P1/P2 proteins

The ribosome has a distinct lateral protuberance called the stalk; in eukaryotes it is formed by the acidic ribosomal P-proteins which are organized as a pentameric entity described as P0-(P1-P2)(2). Bilateral interactions between P0 and P1/P2 proteins have been studied extensively, however, the region on P0 responsible for the binding of P1/P2 proteins has not been precisely defined. Here we report a study which takes the current knowledge of the P0 - P1/P2 protein interaction beyond the recently published information. Using truncated forms of P0 protein and several in vitro and in vivo approaches, we have defined the region between positions 199 and 258 as the P0 protein fragment responsible for the binding of P1/P2 proteins in the yeast Saccharomyces cerevisiae. We show two short amino acid regions of P0 protein located at positions 199-230 and 231-258, to be responsible for independent binding of two dimers, P1A-P2B and P1B-P2A respectively. In addition, two elements, the sequence spanning amino acids 199-230 and the P1A-P2B dimer were found to be essential for stalk formation, indicating that this process is dependent on a balance between the P1A-P2B dimer and the P0 protein.     

Elevated copy number of L-A virus in yeast mutant strains defective in ribosomal stalk

The eukaryotic ribosomal stalk, composed of the P-proteins, is a part of the GTPase-associated-center which is directly responsible for stimulation of translation-factor-dependent GTP hydrolysis. Here we report that yeast mutant strains lacking P1/P2-proteins show high propagation of the yeast L-A virus. Affinity-capture-MS analysis of a protein complex isolated from a yeast mutant strain lacking the P1A/P2B proteins using anti-P0 antibodies showed that the Gag protein, the major coat protein of the L-A capsid, is associated with the ribosomal stalk. Proteomic analysis revealed that the elongation factor eEF1A was also present in the isolated complex. Additionally, yeast strains lacking the P1/P2-proteins are hypersensitive to paromomycin and hygromycin B, underscoring the fact that structural perturbations in the stalk strongly influence the ribosome function, especially at the level of elongation.     

The subcellular distribution of the human ribosomal "stalk" components: P1, P2 and P0 proteins

The ribosomal "stalk" structure is a distinct lateral protuberance located on the large ribosomal subunit in prokaryotic, as well as in eukaryotic cells. In eukaryotes, this ribosomal structure is composed of the acidic ribosomal P proteins, forming two hetero-dimers (P1/P2) attached to the ribosome through the P0 protein. The "stalk" is essential for the ribosome activity, taking part in the interaction with elongation factors.In this report, we have shown that the subcellular distribution of the human P proteins does not fall into standard behavior of regular ribosomal proteins. We have used two approaches to assess the distribution of the P proteins, in vivo experiments with GFP fusion proteins and in vitro one with anti-P protein antibodies. In contrast to standard r-proteins, the P1 and P2 proteins are not actively transported into the nucleus compartment, remaining predominantly in the cytoplasm (the perinuclear compartment). The P0 protein was found in the cytoplasm, as well as in the nucleus; however, the nucleoli were excluded. This protein was scattered around the nuclei, and the distribution might reflect association with the so-called nuclear bodies. This is the first example of r-proteins that are not actively transported into the nucleus; moreover, this might imply that the "stalk" constituents are assembled onto the ribosomal particle at the very last step of ribosomal maturation, which takes part in the cell cytoplasm.     

The amino terminal end determines the stability and assembling capacity of eukaryotic ribosomal stalk proteins P1 and P2

The eukaryotic ribosomal proteins P1 and P2 bind to protein P0 through their N-terminal domain to form the essential ribosomal stalk. A mutational analysis points to amino acids at positions 2 and 3 as determinants for the drastic difference of Saccharomyces cerevisiae P1 and P2 half-life, and suggest different degradation mechanisms for each protein type. Moreover, the capacity to form P1/P2 heterodimers is drastically affected by mutations in the P2β four initial amino acids, while these mutations have no effect on P1β. Binding of P2β and, to a lesser extent, P1β to the ribosome is also seriously affected showing the high relevance of the amino acids in the first turn of the NTD α-helix 1 for the stalk assembly. The negative effect of some mutations on ribosome binding can be reversed by the presence of the second P1/P2 couple in the ribosome, indicating a stabilizing structural influence between the two heterodimers. Unexpectedly, some mutations totally abolish heterodimer formation but allow significant ribosome binding and, therefore, a previous P1 and P2 association seems not to be an absolute requirement for stalk assembly. Homology modeling of the protein complexes suggests that the mutated residues can affect the overall protein conformation.     

Shiga toxin 1 is more dependent on the P proteins of the ribosomal stalk for depurination activity than Shiga toxin 2

Shiga toxins produced by Escherichia coli O157:H7 are responsible for food poisoning and hemolytic uremic syndrome (HUS). The A subunits of Shiga toxins (Stx1A and Stx2A) inhibit translation by depurinating a specific adenine in the large rRNA. To determine if Stx1A and Stx2A require the ribosomal stalk for depurination, their activity and cytotoxicity were examined in the yeast P protein deletion mutants. Stx1A and Stx2A were less toxic and depurinated ribosomes less in a strain lacking P1/P2 on the ribosome and in the cytosol (ΔP2) than in a strain lacking P1/P2 on the ribosome, but containing free P2 in the cytosol (ΔP1). To determine if cytoplasmic P proteins facilitated depurination, Stx1A and Stx2A were expressed in the P0ΔAB mutant, in which the binding sites for P1/P2 were deleted on the ribosome, and P1/P2 accumulated in the cytosol. Stx1A was less toxic and depurinated ribosomes less in P0ΔAB, suggesting that intact binding sites for P1/P2 were critical. In contrast, Stx2A was toxic and depurinated ribosomes in P0ΔAB as in wild type, suggesting that it did not require the P1/P2 binding sites. Depurination of ΔP1, but not P0ΔAB ribosomes increased upon addition of purified P1α/P2βin vitro, and the increase was greater for Stx1 than for Stx2. We conclude that cytoplasmic P proteins stimulate depurination by Stx1 by facilitating the access of the toxin to the ribosome. Although ribosomal stalk is important for Stx1 and Stx2 to depurinate the ribosome, Stx2 is less dependent on the stalk proteins for activity than Stx1 and can depurinate ribosomes with an incomplete stalk better than Stx1.     

Role of the ribosomal stalk components in the resistance of Aspergillus fumigatus to the sordarin antifungals

Aspergillus fumigatus, an important human nosocomial pathogen, is resistant to sordarin derivatives, a new family of antifungals that inhibit protein synthesis by interaction with the EF-2-ribosomal stalk complex. To explore the role of the A. fumigatus ribosome in the resistance mechanism, the fungal stalk proteins were biochemically and genetically characterized and expressed in the sensitive Saccharomyces cerevisiae. Two acidic phosphoproteins homologous to the 12 kDa P1 and P2 proteins described in other organisms were found together with the 34 kDa P0 protein, the third stalk component. The genes encoding each fungal stalk protein were expressed in mutant S. cerevisiae strains lacking the equivalent proteins. Both AfP1 and AfP2 proteins interact with their yeast counterparts of the opposite type and bind to the ribosomal particles in the presence of either the S. cerevisiae or the A. fumigatus P0 protein. The A. fumigatus acidic phosphoproteins did not alter the yeast ribosome sordarin sensitivity. On the contrary, the presence of the fungal P0 induces in vivo and in vitro resistance to sordarin derivatives when present in the yeast ribosome. The mutations A117-->E, P122-->R and G124-->V in A. fumigatus P0 reduce the resistance capacity of the protein. An S. cerevisiae strain with the complete ribosomal stalk of A. fumigatus was obtained, which could be useful for the screening of new antifungals against this pathogenic fungus.     

Asymmetric interactions between the acidic P1 and P2 proteins in the Saccharomyces cerevisiae ribosomal stalk.

The Saccharomyces cerevisiae ribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1alpha, P1beta, P2alpha, and P2beta. The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure. Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1alpha, P0-1beta, P0-2alpha, and P0-2beta. The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth. In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition. An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein. The following associations were found: P0-1alpha.P2beta, P0-1beta.P2alpha, P0-2alpha.P1beta, and P0-2beta.P1alpha. These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1alpha.P2beta and P1beta.P2alpha, which have different structural and functional roles.     

Pentameric organization of the ribosomal stalk accelerates recruitment of ricin a chain to the ribosome for depurination

Ribosome inactivating proteins (RIPs) depurinate a universally conserved adenine in the α-sarcin/ricin loop (SRL) and inhibit protein synthesis at the translation elongation step. We previously showed that ribosomal stalk is required for depurination of the SRL by ricin toxin A chain (RTA). The interaction between RTA and ribosomes was characterized by a two-step binding model, where the stalk structure could be considered as an important interacting element. Here, using purified yeast ribosomal stalk complexes assembled in vivo, we show a direct interaction between RTA and the isolated stalk complex. Detailed kinetic analysis of these interactions in real time using surface plasmon resonance (SPR) indicated that there is only one type of interaction between RTA and the ribosomal stalk, which represents one of the two binding steps of the interaction with ribosomes. Interactions of RTA with the isolated stalk were relatively insensitive to salt, indicating that nonelectrostatic interactions were dominant. We compared the interaction of RTA with the full pentameric stalk complex containing two pairs of P1/P2 proteins with its interaction with the trimeric stalk complexes containing only one pair of P1/P2 and found that the rate of association of RTA with the pentamer was higher than with either trimer. These results demonstrate that the stalk is the main landing platform for RTA on the ribosome and that pentameric organization of the stalk accelerates recruitment of RTA to the ribosome for depurination. Our results suggest that multiple copies of the stalk proteins might also increase the scavenging ability of the ribosome for the translational GTPases.     

Structural differences between Saccharomyces cerevisiae ribosomal stalk proteins P1 and P2 support their functional diversity

The eukaryotic acidic P1 and P2 proteins modulate the activity of the ribosomal stalk but playing distinct roles. The aim of this work was to analyze the structural features that are behind their different function. A structural characterization of Saccharomyces cerevisaie P1 alpha and P2 beta proteins was performed by circular dichroism, nuclear magnetic resonance, fluorescence spectroscopy, thermal denaturation, and protease sensitivity. The results confirm the low structure present in both proteins but reveal clear differences between them. P1 alpha shows a virtually unordered secondary structure with a residual helical content that disappears below 30 degrees C and a clear tendency to acquire secondary structure at low pH and in the presence of trifluoroethanol. In agreement with this higher disorder P1 alpha has a fully solvent-accessible tryptophan residue and, in contrast to P2 beta, is highly sensitive to protease degradation. An interaction between both proteins was observed, which induces an increase in the global secondary structure content of both proteins. Moreover, mixing of both proteins causes a shift of the P1 alpha tryptophan 40 signal, pointing to an involvement of this region in the interaction. This evidence directly proves an interaction between P1 alpha and P2 beta before ribosome binding and suggests a functional complementation between them. On a whole, the results provide structural support for the different functional roles played by the proteins of the two groups showing, at the same time, that relatively small structural differences between the two stalk acidic protein types can result in significant functional changes.     

Solution structure of the natively assembled yeast ribosomal stalk determined by small-angle X-ray scattering

The ribosomal stalk of the 60S subunit has been shown to play a crucial role in all steps of protein synthesis, but its structure and exact molecular function remain an unanswered question. In the present study, we show the low-resolution models of the solution structure of the yeast ribosomal stalk, composed of five proteins, P0-(P1-P2)(2). The model of the pentameric stalk complex determined by small-angle X-ray scattering reveals an elongated shape with a maximum length of 13 nm. The model displays three distinct lobes, which may correspond to the individual P1-P2 heterodimers anchored to the C-terminal domain of the P0 protein.     

Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses

Trichosanthin (TCS) is a type I ribosome-inactivating protein that inactivates ribosome by enzymatically depurinating the A⁴³²⁴ at the α-sarcin/ricin loop of 28S rRNA. We have shown in this and previous studies that TCS interacts with human acidic ribosomal proteins P0, P1 and P2, which constitute the lateral stalk of eukaryotic ribosome. Deletion mutagenesis showed that TCS interacts with the C-terminal tail of P2, the sequences of which are conserved in P0, P1 and P2. The P2-binding site on TCS was mapped to the C-terminal domain by chemical shift perturbation experiments. Scanning charge-to-alanine mutagenesis has shown that K173, R174 and K177 in the C-terminal domain of TCS are involved in interacting with the P2, presumably through forming charge–charge interactions to the conserved DDD motif at the C-terminal tail of P2. A triple-alanine variant K173A/R174A/K177A of TCS, which fails to bind P2 and ribosomal stalk in vitro, was found to be 18-fold less active in inhibiting translation in rabbit reticulocyte lysate, suggesting that interaction with P-proteins is required for full activity of TCS. In an analogy to the role of stalk proteins in binding elongation factors, we propose that interaction with acidic ribosomal stalk proteins help TCS to locate its RNA substrate.     

Tag-mediated fractionation of yeast ribosome populations proves the monomeric organization of the eukaryotic ribosomal stalk structure

The analysis of the not well understood composition of the stalk, a key ribosomal structure, in eukaryotes having multiple 12 kDa P1/P2 acidic protein components has been approached using these proteins tagged with a histidine tail at the C-terminus. Tagged Saccharomyces cerevisiae ribosomes, which contain two P1 proteins (P1 alpha and P1 beta) and two P2 proteins (P2 alpha and P2 beta), were fractionated by affinity chromatography and their stalk composition was determined. Different yeast strains expressing one or two tagged proteins and containing either a complete or a defective stalk were used. No indication of protein dimers was found in the tested strains. The results are only compatible with a stalk structure containing a single copy of each one of the four 12 kDa proteins per ribosome. Ribosomes having an incomplete stalk are found in wild-type cells. When one of the four proteins is missing, the ribosomes do not carry the three remaining proteins simultaneously, containing only two of them distributed in pairs made of one P1 and one P2. Ribosomes can carry two, one or no acidic protein pairs. The P1 alpha/P2 beta and P1beta/P2 alpha pairs are preferentially found in the ribosome, but they are not essential either for stalk assembly or function.     

Interaction map of the Trypanosoma cruzi ribosomal P protein complex (stalk) and the elongation factor 2

The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the translation step of protein synthesis through interaction with the elongation factor 2 (EF‐2). The Trypanosoma cruzi stalk complex is composed of four proteins of about 11 kDa, TcP1α, TcP1β, TcP2α, TcP2β and a fifth TcP0 of about 34 kDa. In a previous work, a yeast two‐hybrid (Y2H) protein–protein interaction map of T. cruzi ribosomal P proteins was generated. In order to gain new insight into the assembly of the stalk, a complete interaction map was generated by surface plasmon resonance (SPR) and the kinetics of each interaction was calculated. All previously detected interactions were confirmed and new interacting pairs were found, such as TcP1β–TcP2α and TcP1β–TcP2β. Moreover P2 but not P1 proteins were able to homo‐oligomerize. In addition, the region comprising amino acids 210–270 on TcP0 was identified as the region interacting with P1/P2 proteins, using Y2H and SPR. The interaction domains on TcP2β were also mapped by SPR identifying two distinct regions. The assembly order of the pentameric complex was assessed by SPR showing the existence of a hierarchy in the association of the different P proteins forming the stalk. Finally, the TcEF‐2 gene was identified, cloned, expressed and refolded. Using SPR analysis we showed that TcEF‐2 bound with similar affinity to the four P1/P2 ribosomal P proteins of T. cruzi but with reduced affinity to TcP0. Copyright © 2010 John Wiley & Sons, Ltd.     

Phosphorylation of Initiation Factor eIF2 in Response to Stress Conditions Is Mediated by Acidic Ribosomal P1/P2 Proteins in Saccharomyces cerevisiae

Eukaryotic cells contain an unusually large cytoplasmic pool of P1/P2 phosphoproteins, which form the highly flexible 60S subunit stalk that is required to interact with and activate soluble translation factors. In cells, cytoplasmic P1/P2 proteins are exchanged for ribosome-bound proteins in a process that can modulate ribosome function and translation. Here, we analysed different S. cerevisiae stalk mutants grown under stress conditions that result in eIF2α phosphorylation. These mutants either lack a cytoplasmic pool of stalk proteins or contain free but not ribosome-bound proteins. Only cells that contain free P1/P2 proteins induce eIF2 phosphorylation in vivo in response to glucose starvation or osmotic stress. Moreover, we show that free S. cerevisiae P1/P2 proteins can induce in vitro phosphorylation of the initiation factor eIF2 by stimulating the autophosphorylation and activation of GCN2 kinase. Indeed, these ribosomal proteins do not stimulate other eIF2α kinases, such as PKR and HRI. P1/P2 and the known GCN2 activator deacylated tRNA compete for stimulating the eIF2α kinase activity of GCN2, although the P1/P2 proteins are considerably more active. These findings reveal a capacity of free cytoplasmic ribosomal stalk components to stimulate eIF2α phosphorylation, which in turn would modulate translation in response to specific forms of stress that may be linked with the previously described regulatory function of the ribosomal stalk.     

Ribosomal cold-adaptation: characterization of the genes encoding the acidic ribosomal P0 and P2 proteins from the Antarctic ciliate Euplotes focardii

Molecular adaptation at low temperature requires specificities represented mainly by modifications in the gene sequence and consequently in the protein primary structure. To characterize the molecular mechanisms responsible for ribosome cold-adaptation, we compared the ribosomal P0 and P2 genes from the Antarctic ciliate Euplotes focardii with homologous genes from mesophilic organisms, including the ciliates Tetrahymena thermophila and non cold-adapted Euplotes species. This analysis revealed the presence of non synonymous mutations unique to E. focardii. In the P0 protein the mutations produced amino acid substitutions that increased the molecular flexibility that may facilitate a conformational adjustment associated with the interaction with the GTPase center of the large subunit rRNA, and increased the hydrophobicity of the region involved in the interaction with P1/P2 heterodimer, probably to keep associated the ribosomal stalk in the cold. In the P2 protein the mutations produced amino acid substitutions that increased the N-terminus flexibility, which may facilitate interactions with P1 protein in the formation of the heterodimer, and reduced the mobility of the C-terminus, to stabilize the stalk during ribosomal activity. Finally, P proteins appeared to be valid markers for investigating the phylogenetic origin of early eukaryotes.     

Structural and functional characterization of the amino terminal domain of the yeast ribosomal stalk P1 and P2 proteins

The essential ribosomal stalk is formed in eukaryotes by a pentamer of two P1–P2 protein heterodimers and the P0 rRNA binding protein. In contrast to the highly stable prokaryotic complex, the P1 and P2 proteins in the eukaryotic stalk undergo a cyclic process of assembly and disassembly during translation that seems to modulate the ribosome activity. To better understand this process, the regions of the Saccharomyces cerevisiae P1α and P2β proteins that are directly involved in heterodimer formation and ribosome binding have been characterized using a series of P1α/P2β chimeras. The region required for a stable interaction with the ribosome is formed by the first three predicted α-helices in the N-terminal domain of both proteins. The same region is required for heterodimer formation in P2β but the third helix is dispensable for this association in P1α. It seems, therefore, that stable ribosome binding is more structurally demanding than heterodimerization. A fourth predicted α-helix in the N-terminal domain of P1α and P2β appears not to be involved in the assembly process but rather, it contributes to the conformation of the proteins by apparently restricting the mobility of their C-terminal domain and paradoxically, by reducing their activity. In addition, the study of P1/P2 chimeras showed that the C-terminal domains of these two types of protein are functionally identical and that their protein specificity is exclusively determined by their N-terminal domains.