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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

Conformational changes induced in the Saccharomyces cerevisiae GTPase-associated rRNA by ribosomal stalk components and a translocation inhibitor

The yeast ribosomal GTPase associated center is made of parts of the 26S rRNA domains II and VI, and a number of proteins including P0, P1α, P1β, P2α, P2β and L12. Mapping of the rRNA neighborhood of the proteins was performed by footprinting in ribosomes from yeast strains lacking different GTPase components. The absence of protein P0 dramatically increases the sensitivity of the defective ribosome to degradation hampering the RNA footprinting. In ribosomes lacking the P1/P2 complex, protection of a number of nucleotides is detected around positions 840, 880, 1100, 1220–1280 and 1350 in domain II as well as in several positions in the domain VI α-sarcin region. The protection pattern resembles the one reported for the interaction of elongation factors in bacterial systems. The results exclude a direct interaction of these proteins with the rRNA and are compatible with an increase in the ribosome affinity for EF-2 in the absence of the acidic P proteins. Interestingly, a sordarin derivative inhibitor of EF-2 causes an opposite effect, increasing the reactivity in positions protected by the absence of P1/P2. Similarly, a deficiency in protein L12 exposes nucleotides G1235, G1242, A1262, A1269, A1270 and A1272 to chemical modification, thus situating the protein binding site in the most conserved part of the 26S rRNA, equivalent to the bacterial protein L11 binding site.     

Phosphorylation of the yeast ribosomal stalk. Functional effects and enzymes involved in the process

The ribosomal stalk is directly involved in the interaction of the elongation factors with the ribosome during protein synthesis. The stalk is formed by a complex of five proteins, four small acidic polypeptides and a larger protein which directly interacts with the rRNA at the GTPase center. In eukaryotes the acidic components correspond to the 12-kDa P1 and P2 proteins, and the RNA binding component is the P0 protein. All these proteins are found phosphorylated in eukaryotic organisms, and previous in vitro data suggested this modification was involved in the activity of this structure. Results from mutational studies have shown that phosphorylation takes place at a serine residue close to the carboxy end of the P proteins. Modification of this serine residue does not affect the formation of the stalk and the activity of the ribosome in standard conditions but induces an osmoregulation-related phenotype at 37 degrees C. The phosphorylatable serine is part of a consensus casein kinase II phosphorylation site. However, although CKII seems to be responsible for part of the stalk phosphorylation in vivo, it is probably not the only enzyme in the cell able to perform this modification. Five protein kinases, RAPI, RAPII and RAPIII, in addition to the previously reported CKII and PK60 kinases, are able to phosphorylate the stalk proteins. A comparison of the five enzymes shows differences among them that suggest some specificity regarding the phosphorylation of the four yeast acidic proteins. It has been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins. All the data suggest that although phosphorylation is not involved in the interaction of the acidic P proteins with the ribosome, it can affect the ribosome activity and might participate in a possible ribosome regulatory mechanism.     

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.     

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.     

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.     

On the role of cyclic AMP-independent protein kinases in the modification of yeast ribosomal proteins in vivo

Two proteins of yeast 40S ribosome subunit and four proteins of the 60S ribosome subunit were labelled in vivo with [32P]orthophosphate. Five of these proteins were phosphorylated by protein kinase 3, an enzyme which is cyclic AMP-independent and uses ATP and GTP as phosphoryl donors. Two proteins, belonging to the 60S ribosome subunit were phosphorylated by another, highly specific, cyclic AMP-independent protein kinase 1 B. Both in vivo and in vitro the most extensively phosphorylated protein species were acidic proteins, L44, L45 (according to the nomenclature of Kruiswijk & Planta, Molec. Biol. Rep., 1, 409-415, 1974) possibly corresponding to bacterial L7 and L12 proteins. The 40S ribosomal protein, S9, analogous to mammalian S6 protein, was phosphorylated in vivo but was not phosphorylated in vitro by either of the cyclic AMP-independent protein kinases. The obtained results clearly indicate that cyclic AMP-independent yeast protein kinases might be involved in the modification in vivo of some ribosomal proteins, in particular of the strongly acidic proteins of 60S ribosome subunit.     

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.     

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.     

Protein kinases CKI and CKII are implicated in modification of ribosomal proteins of the yeast Trichosporon cutaneum

Phosphorylation of acidic ribosomal proteins P1/P2-P0 is a common phenomenon in eukaryotic organisms. It was found previously that in Trichosporon cutaneum, unlike in other yeast species, in addition to the two acidic ribosomal proteins, two other proteins of 15 kDa and 19 kDa of the small ribosomal subunit were phosphorylated. Here we describe two protein kinases: CKI and CKII, which are engaged in the modification of T. cutaneum ribosomal proteins. The acidic ribosomal proteins and the protein of 19 kDa were modified by CKII associated with ribosomes, while the protein of 15 kDa was modified by CKI. Protein kinase CKI was purified from cell-free extract (CKIC) and from ribosomal fraction (CKIR). The molecular mass of CKIC was established at 33 kDa while that of CKIR at 35-37 kDa. A protein of 40 kDa copurified with CKIR but not CKIC. Heparin significantly increased 40 kDa protein phosphorylation level by CKIR. Microsequencing analysis revealed the presence of CKI recognition motifs in the N-terminal fragment of the 40 kDa protein.     

The gene and the primary structure of acidic ribosomal protein A0 from yeast Saccharomyces cerevisiae which shows partial homology to bacterial ribosomal protein L10

Eukaryotic ribosomes contain an acidic ribosomal protein of about 38 kDa which shows immunological cross-reactivity with the 13 kDa-type acidic ribosomal proteins that are related to L7/L12 of bacterial ribosomes. By using a cDNA clone for 38 kDa-type acidic ribosomal protein A0 from the yeast Saccharomyces cerevisiae, we have cloned a genomic DNA encoding A0 and determined the sequence of 1,614 nucleotides including about 500 nucleotides in the 5'-flanking region. The gene lacks introns and possesses two boxes homologous to upstream activation sequences (UASrpg) in the 5'-flanking region. The amino acid sequence of A0 deduced from the nucleotide sequence shows that A0 shares a highly similar carboxyl-terminal region of about 40 amino acids in length with 13 kDa-type acidic ribosomal proteins, including an identical carboxyl-terminal, DDDMGFGLFD. In the amino-terminal region A0 contains an arginine-rich segment which shows a low but distinct similarity to that of bacterial ribosomal protein L10 through which L10 is thought to bind to 23S rRNA. On the other hand, the carboxyl-terminal half of A0 is enriched with hydrophobic amino acid residues including four pairs of phenylalanine residues which are all conserved in a human homologue.