Functional complementation of yeast ribosomal P0 protein with Plasmodium falciparum P0

A complex of three phosphoproteins (P0, P1 and P2) constitutes the stalk region at the GTPase center of the eukaryotic large ribosomal subunit, amongst which the protein P0 plays the most crucial role. Earlier studies have shown the functional complementation of the conditional P0-null mutant of Saccharomyces cerevisiae (W303dGP0) with orthologous P0 genes from fungal and mammalian organisms, but not the protozoan parasite Leishmania infantum. In this paper we show that the PfP0 gene from the protozoan malaria parasite Plasmodium falciparum can functionally complement the conditional P0-null W303dGP0 mutant of S. cerevisiae. Unlike the above orthologous genes, PfP0 gene could also rescue the D67dGP0 strain, which in addition to being a conditional null for ScP0 gene, is a null-mutant for both ScP1alpha and beta genes. However, under stress conditions such as high temperature, salt and osmolarity, PfP0 gene could not rescue D67dGP0 strain. Ribosomes purified from W303dGP0 carrying PfP0 gene did not contain ScP1 protein, indicating a lack of binding of ScP1 to PfP0 protein. Yeast 2-hybrid analysis further confirmed the lack of binding of ScP1 to PfP0 protein. The polymerizing activities of ribosomes with ScP0 or PfP0 protein, in the absence of ScP1 protein, were found to be about 40-45% that of ribosomes with all the yeast P-proteins. In its sensitivity to the inhibitor sordarin, PfP0 was similar to the P0 protein from the fungus Aspergillus fumigatus. These results indicate a closer functional relationship of P. falciparum P0 gene to fungal P0 genes.     

The ribosomal P-proteins of the medfly Ceratitis capitata form a heterogeneous stalk structure interacting with the endogenous P-proteins, in conditional P0-null strains of the yeast Saccharomyces cerevisiae

The genes encoding the ribosomal P-proteins CcP0, CcP1 and CcP2 of Ceratitis capitata were expressed in the conditional P0-null strains W303dGP0 and D67dGP0 of Saccharomyces cerevisiae, the ribosomes of which contain either standard amounts or are totally deprived of the P1/P2 proteins, respectively. The presence of the CcP0 protein restored cell viability but reduced the growth rate. In the W303CcP0 strain, all four acidic yeast proteins were found on the ribosomes, but in notably less quantity, while a preferable binding of the YP1α/YP2β pair was established. In the absence of the endogenous P1/P2 proteins in the D67CcP0 strain, the complementation capacity of the CcP0 protein was considerably reduced. The simultaneous expression of the three medfly genes resulted in alterations of the stalk composition: both the CcP1 and CcP2 proteins were found on the particles substituting the YP1α and YP2α proteins, respectively, but their presence did not alter the growth rate, except in the case of the YP1α/β defective strain, where a helping effect on the binding of the YP2α and YP2β proteins on the ribosomes was confirmed. Therefore, the medfly ribosomal P-proteins complement the yeast P-protein deficient strains forming an heterogeneous ribosomal stalk, which, however, is not functionally equivalent to the endogenous one.     

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.     

Mass spectrometry of ribosomes from Saccharomyces cerevisiae: implications for assembly of the stalk complex

The acidic ribosomal P proteins form a distinct protuberance on the 60 S subunit of eukaryotic ribosomes. In yeast this structure is composed of two heterodimers (P1alpha-P2beta and P1beta-P2alpha) attached to the ribosome via P0. Although for prokaryotic ribosomes the isolation of a pentameric stalk complex comprising the analogous proteins is well established, its observation has not been reported for eukaryotic ribosomes. We used mass spectrometry to examine the composition of the stalk proteins on ribosomes from Saccharomyces cerevisiae. The resulting mass spectra reveal a noncovalent complex of mass 77,291 +/- 7 Da assigned to the pentameric stalk. Tandem mass spectrometry confirms this assignment and is consistent with the location of the P2 proteins on the periphery of the stalk complex, shielding the P1 proteins, which in turn interact with P0. No other oligomers are observed, confirming the specificity of the pentameric complex. At lower m/z values the spectra are dominated by individual proteins, largely from the stalk complex, giving rise to many overlapping peaks. To define the composition of the stalk proteins in detail we compared spectra of ribosomes from strains in which genes encoding either or both of the interacting stalk proteins P1alpha or P2beta are deleted. This enables us to define novel post-translational modifications at very low levels, including a population of P2alpha molecules with both phosphorylation and trimethylation. The deletion mutants also reveal interactions within the heterodimers, specifically that the absence of P1alpha or P2beta destabilizes binding of the partner protein on the ribosome. This implies that assembly of the stalk complex is not governed solely by interactions with P0 but is a cooperative process involving binding to partner proteins for additional stability on the ribosome.     

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.     

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.     

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.     

The amino terminal domain from Mrt4 protein can functionally replace the RNA binding domain of the ribosomal P0 protein

In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein.     

In vivo analysis of the acidic ribosomal proteins BmP1 and BmP2 of the silkworm Bombyx mori in the yeast Saccharomyces cerevisiae

In the silkworm Bombyx mori the ribosomal stalk P-protein family consists of two low MW acidic proteins, BmP1 and BmP2, and of one higher MW protein, BmP0, as shown by electrophoretical and immunoblotting western blot analysis of purified ribosomes. Treatment of ribosomes with alkaline phosphatase followed by electrofocusing shifted the isoelectric points to higher pH, implying phosphorylation of the proteins. The cDNAs encoding BmP1 and BmP2 proteins were constructed and expressed in the Saccharomyces cerevisiae mutant strains defective in either the endogenous P1 or P2 proteins. The recombinant silkworm proteins could complement the absence of the homologous yeast proteins and were incorporated to the ribosomes of the transformed strains, helping the binding of the remaining endogenous acidic proteins, present in the cytoplasm in different extent. Thus, BmP1 was able to replace YP1alpha, preferentially binding YP2beta to the ribosome, while BmP2 replaced both yeast P2 proteins and induced the binding of both YP1alpha and YP1beta.     

Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently.     

八肋游仆虫酸性核糖体磷酸化蛋白P1有2个亚型

真核生物酸性核糖体磷酸化蛋白(P0、P1、P2)位于核糖体60S大亚基上,它们在核糖体上共同组成一个向外侧凸出的五聚体的柄状复合物［P0·(P1·P2)2］,该复合物在蛋白质合成延伸过程中起着重要作用．为了探讨单细胞真核生物核糖体柄状复合物的组成形式及在蛋白质合成中的作用,对八肋游仆虫（Euplotes octocarinatus）的P1进行了研究．通过生物信息学方法,分析八肋游仆虫基因组及转录组数据,找到2个酸性核糖体蛋白P1基因,从DNA 和cDNA中都扩增到这2个P1基因,表明八肋游仆虫酸性核糖体磷酸化蛋白P1确实存在2个亚型. 将2个基因克隆后分别构建重组表达质粒pET28a-P1A和pGEX-6P-1-P1B,在大肠杆菌BL21中获得高效表达.经镍柱和GST柱亲和层析后,获得较高纯度的八肋游仆虫酸性核糖体蛋白EoP1A和EoP1B,表达产物经Western印迹检测为阳性．Pull-down分析了EoP1A和EoP1B之间的相互作用．结果表明,游仆虫酸性核糖体磷酸化蛋白P1的2个亚型EoP1A和EoP1B之间存在相互作用.     

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.     

Selective blockade of trypanosomatid protein synthesis by a recombinant antibody anti-Trypanosoma cruzi P2β protein

The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.     

Characterization of a novel protein inhibitor of protein kinases specific to acidic ribosomal proteins

The ribosomal stalk composed of acidic P1/P2 proteins and protein P0 is involved directly in the interaction of the elongation factors and mRNAs with the ribosome during protein synthesis. All P proteins are found to be phosphorylated in eucaryotic organisms. In Saccharomyces cerevisiae five different cAMP-independent protein kinases phosphorylating P proteins have been identified and characterized. In contrast to many other protein kinases, relatively little is known about inhibitors of these enzymes. A new protein inhibitor of protein kinases has been purified and characterized. It is a small (18.5 kDa) and acidic (pI = 4.2) protein with high inhibitory potency for PK60S and CK 2. The inhibitor is competitive with respect to protein substrates with Ki values in the range of approximately 6.5 microM for PK60S and approximately 22 microM for CK 2.     

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.     

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.     

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.     

Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts.

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.     

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.