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.     

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.     

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.     

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.     

Different roles of P1 and P2 Saccharomyces cerevisiae ribosomal stalk proteins revealed by cross-linking

The stalk is an essential domain of the large ribosomal subunit formed by a complex of a set of very acidic proteins bound to a core rRNA binding component. While in prokaryotes there is only one type acidic protein, L7/12, two protein families are found in eukaryotes, phosphoproteins P1 and P2, which presumably have different roles. To search for differences zero-length cross-linking by S-S bridge formation was applied using Saccharomyces cerevisiae mutant P1 and P2 proteins carrying single cysteine residues at various positions. The results show a more exposed location of the N-terminal domain of the P2 proteins, which in contrast to P1, can be found as dimers when the Cys is introduced in this domain. Similarly, the Cys containing C-terminal domain of mutant P2 proteins shows a notable capacity to form cross-links with other proteins, which is considerably lower in the P1 type. On the other hand, mutation at the conserved C-domain of protein P0, the eukaryotic stalk rRNA binding component, results in removal of about 14 terminal amino acids. Protein P2, but not P1, protects mutant P0 from this truncation. These results support a eukaryotic stalk structure in which P1 proteins are internally located with their C-terminals having a restricted reactivity while P2 proteins are more external and accessible to interact with other cellular components.     

Acquisition of a stable structure by yeast ribosomal P0 protein requires binding of P1A-P2B complex: in vitro formation of the stalk structure

Saccharomyces cerevisiae ribosomal stalk consists of five proteins: P0 protein, with molecular mass of 34 kDa, and four small, 11 kDa, P1A, P1B, P2A and P2B acidic proteins, which form a pentameric complex P0-(P1A-P2B)/(P1B-P2A). This structure binds to a region of 26S rRNA termed GTPase-associated domain and plays a crucial role in protein synthesis. The consecutive steps leading to the formation of the stalk structure have not been fully elucidated and the function of individual P-proteins in the assembling of the stalk and protein synthesis still remains elusive. We applied an integrated approach in order to examine all the P-proteins with respect to stalk assembly. Several in vitro methods were utilized to mimic protein self-organization in the cell. Our efforts resulted in reconstitution of the whole recombinant stalk in solution as well as on the ribosomal particle. On the basis of our analysis, it can be inferred that the P1A-P2B protein complex may be regarded as the key element in stalk formation, having structural and functional importance, whereas P1B-P2A protein complex is implicated in regulation of stalk function. The mechanism of quaternary structure formation could be described as a sequential co-folding/association reaction of an oligomeric system with P0-(P1A-P2B) protein complex as an essential element in the acquisition of a stable quaternary structure of the ribosomal stalk. On the other hand, the P1B-P2A complex is not involved in the cooperative stalk formation and our results indicate an increased rate of protein synthesis due to the latter protein pair.     

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.     

Chagas disease: a homology model for the three-dimensional structure of the Trypanosoma cruzi ribosomal P0 antigenic protein

Ribosomal P proteins form a “stalk” complex in the large subunit of the ribosomes. In Trypanosoma cruzi, the etiological agent of Chagas disease, the complex is formed by five P protein members: TcP0, TcP1α, TcP1β, TcP2α and TcP2β. The TcP0 protein has 34 kDa, and TcP1 and TcP2 proteins have 10 kDa. The structure of T. cruzi P0 and the stalk complex TcP0–TcP1α–TcP1β–TcP2α–TcP2β have not been solved to date. In this work, we constructed a three-dimensional molecular model for TcP0 using homology modeling as implemented in the MODELLER 9v12 software. The model was constructed using different templates: the X-ray structures of the protein P0 from Pirococcus horikoshii, a segment from the Danio renio Ca⁺²/K⁺ channel and the C-terminal peptide (C13) from T. cruzi ribosomal P2 protein; the Cryo-EM structure of Triticum aestivum P0 protein and the NMR structure of Homo sapiens P1 ribosomal protein. TcP0 has a 200-residue-long N-terminal, which is an α/β globular stable domain, and a flexible C-terminal, 120-residue-long domain. The molecular surface electrostatic potential and hydrophobic surface were calculated. The surface properties are important for the C-terminal's antigenic properties. They are also responsible for P0-specific binding to RNA26S and the binding to the P1–P2 proteins. We explored and identified protein interactions that may be involved in conformational stability. The structure proposed in this work represents a first structural report for the TcP0 protein.     

The RNA interacting domain but not the protein interacting domain is highly conserved in ribosomal protein P0

Protein P0 interacts with proteins P1alpha, P1beta, P2alpha, and P2beta, and forms the Saccharomyces cerevisiae ribosomal stalk. The capacity of RPP0 genes from Aspergillus fumigatus, Dictyostelium discoideum, Rattus norvegicus, Homo sapiens, and Leishmania infantum to complement the absence of the homologous gene has been tested. In S. cerevisiae W303dGP0, a strain containing standard amounts of the four P1/P2 protein types, all heterologous genes were functional except the one from L. infantum, some of them inducing an osmosensitive phenotype at 37 degrees C. The polymerizing activity and the elongation factor-dependent functions but not the peptide bond formation capacity is affected in the heterologous P0 containing ribosomes. The heterologous P0 proteins bind to the yeast ribosomes but the composition of the ribosomal stalk is altered. Only proteins P1alpha and P2beta are found in ribosomes carrying the A. fumigatus, R. norvegicus, and H. sapiens proteins. When the heterologous genes are expressed in a conditional null-P0 mutant whose ribosomes are totally deprived of P1/P2 proteins, none of the heterologous P0 proteins complemented the conditional phenotype. In contrast, chimeric P0 proteins made of different amino-terminal fragments from mammalian origin and the complementary carboxyl-terminal fragments from yeast allow W303dGP0 and D67dGP0 growth at restrictive conditions. These results indicate that while the P0 protein RNA-binding domain is functionally conserved in eukaryotes, the regions involved in protein-protein interactions with either the other stalk proteins or the elongation factors have notably evolved.     

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.     

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.     

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.     

Structural properties of the human acidic ribosomal P proteins forming the P1-P2 heterocomplex

The ribosome has a morphologically distinct structural feature called the stalk, recognized as a vital element for its function. The ribosomal P proteins constitute the main part of the eukaryotic ribosomal stalk, forming a pentameric structure P0-(P1-P2)(2). The group of P1/P2 proteins in eukaryotes is very diverse, and in spite of functional and structural similarities they do not fully complement one another, probably constituting an adaptive feature of the ribosome from a particular species to diverse environmental conditions. The functional differences among the P1/P2 proteins were analysed in vivo several times; however, a thorough molecular characterization was only done for the yeast P1/P2 proteins. Here, we report a biophysical analysis of the human P1 and P2 proteins, applying mass spectrometry, CD and fluorescence spectroscopy, cross-linking and size exclusion chromatography. The human P1/P2 proteins form stable heterodimer, as it is the case for P1/P2 from yeast. However, unlike the yeast complex P1A-P2B, the human P1-P2 dimer showed a three-state transition mechanism, suggesting that an intermediate species may exist in solution.     

The acidic protein binding site is partially hidden in the free Saccharomyces cerevisiae ribosomal stalk protein P0

The ribosomal stalk is essential for translation; however, its overall structure is poorly understood. Characterization of the region involved in the interactions between protein P0 and the 12 kDa acidic proteins P1 and P2 is fundamental to understand the assembly and function of this structure in the eukaryotic ribosome. The acidic protein content is important for the ribosome efficiency and affects the translation of specific mRNAs. By usage of a series of progressively truncated fragments of protein P0 in the two-hybrid test, a region between positions 213 and 250 was identified as the minimal protein part able to interact with the acidic proteins. Extensions at either end affect the binding capacity of the fragment either positively or negatively depending on the number of added amino acids. Deletions inside the binding region confirm its in vivo relevance since they drastically reduce the P0 interacting capacity with the 12 kDa acidic proteins, which are severely reduced in the ribosome when the truncated protein is expressed in the cell. Moreover, recombinant His-tagged P0 fragments containing the binding site and bound to Ni(2+)-NTA columns can form a complex with the P1 and P2 proteins, which is able to bind elongation factor EF2. The results indicate the existence of a region in P0 that specifically interacts with the 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 ribosomal stalk.     

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.     

Cloning and purification of protein kinase CK2 recombinant alpha and beta subunits from the Mediterranean fly Ceratitis capitata

The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (β) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2β subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2β subunit, that also possess the conserved features of regulatory CK2β subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2β proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.     

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.     

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.     

Yeast ribosomal stalk heterogeneity in vivo shown by two-photon FCS and molecular brightness analysis

The stalk of Saccharomyces cerevisiae ribosomes contains, on average, five distinct proteins, namely P0 and four acidic proteins, P1α, P1β, P2α, and P2β. Each ribosome contains only one copy of P0, but the distribution of the acidic proteins among the ribosome population in vivo has not been determined. Using two-photon fluorescence correlation spectroscopy and scanning FCS, on cells expressing EGFP-tagged P0, P1, and P2 proteins, we show, with brightness analysis, that individual yeast ribosomes in vivo are compositionally heterogeneous in regard to P1α, P1β, P2α, and P2β. These results are relevant to the hypothesis, based on in vitro studies, that the overall cellular pattern of expressed proteins can be determined by the distribution of the stalk proteins among the ribosome population.     

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.