RNA binding strategies of ribosomal proteins.

Structures of a number of ribosomal proteins have now been determined by crystallography and NMR, though the complete structure of a ribosomal protein-rRNA complex has yet to be solved. However, some ribosomal protein structures show strong similarity to well-known families of DNA or RNA binding proteins for which structures in complex with cognate nucleic acids are available. Comparison of the known nucleic acid binding mechanisms of these non-ribosomal proteins with the most highly conserved surfaces of similar ribosomal proteins suggests ways in which the ribosomal proteins may be binding RNA. Three binding motifs, found in four ribosomal proteins so far, are considered here: homeodomain-like alpha-helical proteins (L11), OB fold proteins (S1 and S17) and RNP consensus proteins (S6). These comparisons suggest that ribosomal proteins combine a small number of fundamental strategies to develop highly specific RNA recognition sites.     

Alpha-sarcin cleaves ribosomal RNA at the alpha-sarcin site in the absence of ribosomal proteins

alpha-Sarcin is capable of specifically cleaving the single phosphodiester bond in the "alpha-sarcin site" of both Escherichia coli and Saccharomyces cerevisiae large rRNAs in the absence of ribosomal proteins. With both sources of rRNA, the rate of cleavage was comparable with and without ribosomal proteins but more complete cleavage was observed in the absence of ribosomal proteins. These observations contrast with earlier findings and indicate that ribosomal proteins are not essential to the unique specificity of the cleavage of rRNA by alpha-sarcin.     

Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins: improved resolution with Triton X-100

The nonionic detergent Triton X-100 binds in varying proportions to specific ribosomal proteins and decreases the relative mobility of these proteins during electrophoresis. When Triton X-100 binds to these ribosomal proteins in the first-dimension gel, the resolution of the ribosomal proteins in the second-dimension gel pattern is greatly improved. Maximum binding of Triton X-100 to the ribosomal proteins is dependent on pH, urea concentration, and the complete reduction of cysteine and methionine. After first-dimension electrophoresis the Triton X-100 in the gel does not interfere with the binding of sodium dodecyl sulfate to the ribosomal proteins and the molecular weight of these proteins can still be estimated directly from the second-dimension slab gel.     

The social life of ribosomal proteins

Ribosomal proteins hold a unique position in biology because their function is so closely tied to the large rRNAs of the ribosomes in all kingdoms of life. Following the determination of the complete crystal structures of both the large and small ribosomal subunits from bacteria, the functional role of the proteins has often been overlooked when focusing on rRNAs as the catalysts of translation. In this review we highlight some of the many known and important functions of ribosomal proteins, both during translation on the ribosome and in a wider context.     

Mutational alterations in 50 proteins of the Escherichia coli ribosome.

Summary A strain of Escherichia coli, VT, which spontaneously gives rise to mutations in many ribosomal proteins, has been used in conjunction with chemical mutagenesis and varying the subsequent incubation temperature to select mutants which have alterations in every ribosomal protein amenable to analysis of 70 S proteins on two-dimensional polyacrylamide gels under standard conditions. Alterations have been detected in 50 ribosomal proteins, namely in 20 from the small and in 30 from the large subunit. This is the most complete set of mutants with altered ribosomal proteins described so far. The difficulty until recently in obtaining mutations in most ribosomal proteins arises not because they are lethal, as has often been supposed, but because of the lack of a suitable selection heretofore.Communicated by E. Bautz     

Characterization of cDNA encoding a L37a ribosomal protein from Taenia crassiceps and its potential use in phylogenetic reconstructions

In this study, we characterized for the first time the complete sequence of a L37a cDNA from a cestode specie: Taenia crassiceps. A phylogenetic analysis of L37a ribosomal proteins from distant animal species is presented and the potential use of such proteins in molecule-based phylogeny is discussed.     

A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.     

Dissociation of yeast 80 S ribosomes by chaotropic salts

Exposure of yeast 80 S ribosomes to chaotropic salts such as NaClO₄ or NaSCN at concentrations as low as 0.4 M resulted in complete dissociation and subsequent aggregation of the ribosomal proteins. However, under similar conditions, both NaCl and NaBr did not cause dissociation and aggregation. The protein precipitate obtained by exposing the ribosomes to 0.5 M NaClO₄ was free of any rRNA contamination as judged by ultraviolet-absorption analysis. Comparison of the two-dimensional polyacrylamide gel electrophoretic analysis of the above ribosomal protein precipitate with that ribosomal proteins isolated by the standard acetic acid extraction procedure revealed that the protein precipitate contained all the ribosomal proteins. Based on these results, a simple method for the isolation of total ribosomal proteins and rRNA under mild, nondenaturing conditions is proposed. A possible mechanism for the dissociation of proteins from the ribosome by chaotropic salts is also discussed.     

Identification of four proteins from the small subunit of the mammalian mitochondrial ribosome using a proteomics approach

Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two-dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in-gel Endoprotease Lys-C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in-gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico. The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases. Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins are homologs to Escherichia coli S9 and S5 proteins. The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi.     

Molecular interactions of ribosomal components

Summary Five of the 30S ribosomal proteins from E. coli were tested for their ability to bind to 16S ribosomal RNA. Only one of these, S15, can form a complex with the RNA. Quantitative measurements as well as competition experiments show that the RNA binding site for the attachment of S15 is specific for this protein.These experiments complete our analysis of all 21 of the 30S ribosomal proteins. Five of these have now been shown to form a site-specific complex with 16S RNA. These are S4, S7, S8, S15 and S20. The relationship of these data to the assembly and structure of the ribosome are discussed.     

The primary structures of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui

The complete amino acid sequences of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui were determined. The sequences were established by manual sequencing of peptides produced with several proteases as well as by cleavage with dilute HCl. Proteins L16, L23 and L33 consist of 119, 154 and 69 amino acid residues, and their molecular masses are 13538, 16812 and 7620 Da, respectively. The comparison of their sequences with those of ribosomal proteins from other organisms revealed that L23 and L33 are related to eubacterial ribosomal proteins from Escherichia coli and Bacillus stearothermophilus, while protein L16 was found to be homologous to a eukaryotic ribosomal protein from yeast. These results provide information about the special phylogenetic position of archaebacteria.     

Sequence homologies amongE. coli ribosomal proteins: Evidence for evolutionarily related groupings and internal duplications

Summary The complete or partial sequences of 47E. coli ribosomal proteins described in the literature have been examined by computerized search and matching programs. In contrast to results previously reported by other investigators, sequence homologies were uncovered among some of these ribosomal proteins that are well beyond statistical expectations. Moreover, alignments of the most strongly homologous sequences suggested the existence of a network of family groupings. Several of these proteins also exhibit internal homologies, indicating that they have been elongated by a series of tandem duplications.     

The Escherichia coli 30S ribosomal subunit; an optimized three-dimensional fit between the ribosomal proteins and the 16S RNA.

We have generated a computerized fit between the 3-dimensional map of the E.coli 30S ribosomal proteins, as determined by neutron scattering, and the recently published 3-dimensional model for the 16S RNA. To achieve this, the framework of coordinates for RNA-protein cross-link sites on the phosphate backbone in the RNA model was related to the corresponding framework of coordinates for the mass centres of the proteins by a least squares fitting procedure. The resulting structure, displayed on a computer graphics system, gives the first complete picture of the E.coli 30S ribosomal subunit showing both the proteins and the double-helical regions of the RNA. The root mean square distance between cross-link sites and protein centres is 32 A. The position of the mass centre of the combined double-helical regions was calculated from the model and compared with the position of the mass centre of the complete set of proteins. The two centres are displaced relative to one another by 20 A in the model structure, in good agreement with the experimental value of 25 A found by neutron scattering.     

Extraction of proteins from Saccharomyces cerevisiae ribosomes under nondenaturing conditions

The differential sensitivity of ribosomal proteins to removal by salts has been studied. Proteins were extracted from the large and small subunits of cytoplasmic ribosomes from Saccharomyces cerevisiae by washing the individual subunits with a series of solutions containing increasing concentrations of NH4Cl (0.74-3.56 M) for a defined time (20 min) at 0 degrees C. The molar ratio of magnesium to ammonium ions of 1:40 was maintained to protect the ribosomal subparticles from complete disassembly. Proteins extracted under each salt condition were analyzed for composition by two-dimensional polyacrylamide gel electrophoresis. The relative quantity of each protein was determined. Most proteins were not removed from the ribosomal particle completely by any one condition, but were preferentially enriched in a single fraction. Whereas most proteins could be solubilized, several proteins remained predominantly or exclusively with the final core particle. The kinetics of protein release from both subunits at a single NH4Cl concentration (0.74 M) were also studied. Release of protein was time dependent, i.e., longer extraction generally removed more of the same proteins. However, prolonged treatment (240 min) of subunits, even at the same salt concentration, resulted in removal of additional species of proteins in varying amounts. Among the ribosomal RNA species, only the 5 S RNA species was released from the ribosomal particles upon treatment.     

Relationship between rates of synthesis and intracellular distribution of ribosomal proteins during oogenesis in the mouse

Ribosomal proteins are synthesized continuously in nonequimolar amounts during oogenesis in the mouse (M. J. LaMarca and P. M. Wassarman, 1979, Develop. Biol. 73, 103), even though ribosomal proteins are found in equimolar amounts in ribosomes. In this report, the distribution of newly synthesized ribosomal proteins between the cytoplasm and germinal vesicle (nucleus) of fully grown mouse oocytes has been examined. As compared to total newly synthesized protein, ribosomal proteins were found to be highly concentrated in the oocyte's germinal vesicle. Furthermore, an inverse relationship was found between rates of synthesis of individual ribosomal proteins and percentages of newly synthesized ribosomal proteins associated with germinal vesicles. As a result of this relationship, the amounts of newly synthesized ribosomal proteins associated with germinal vesicles approximated an equimolar situation. Even in the presence of actinomycin D, oocytes continued to synthesize ribosomal proteins which were found associated with germinal vesicles in amounts similar to those observed in the absence of the drug. These results suggest that, although synthesis of ribosomal proteins by mouse oocytes is not coordinately regulated, a post-translational mechanism exists for adjusting the stoichiometry of these proteins within the oocyte's germinal vesicle; this mechanism apparently is not dependent upon concomitant ribosomal-RNA synthesis.     

Translational machinery of channel catfish: II. Complementary DNA and expression of the complete set of 47 60S ribosomal proteins

Ribosomal protein genes have become widely used as markers for phylogenetic studies and comparative genomics, but they have not been available in fish. We have cloned and sequenced a complete set of all 47 60S ribosomal protein cDNAs from channel catfish (Ictalurus punctatus), of which 43 included the complete protein encoding regions. Most ribosomal protein mRNAs in channel catfish are highly similar to their mammalian counterparts. However, L4, L14, and L29 are significantly shorter in channel catfish than in mammals due to deletions in the 3' end of the gene. Two distantly related L5 cDNAs, L5a and L5b, were found in channel catfish. L5a is more similar to L5 in other vertebrates, while L5b showed significant levels of divergence, suggesting independent evolution of the two L5-encoding genes. The 47 ribosomal protein genes are generally highly expressed and together account for 11-14% of overall gene expression, depending on the tissues. Expression levels were highly variable both within a single tissue among different ribosomal protein genes, and among tissues with regard to a single ribosomal protein gene. Strong tissue preference expression was also observed for some ribosomal proteins. This set of ribosomal protein gene sequences represents one of the most complete sets from any single organism and will aid in fish phylogenetic and comparative genomic studies.     

Two C or not two C: recurrent disruption of Zn-ribbons, gene duplication, lineage-specific gene loss, and horizontal gene transfer in evolution of bacterial ribosomal proteins

Background

Ribosomal proteins are encoded in all genomes of cellular life forms and are, generally, well conserved during evolution. In prokaryotes, the genes for most ribosomal proteins are clustered in several highly conserved operons, which ensures efficient co-regulation of their expression. Duplications of ribosomal-protein genes are infrequent, and given their coordinated expression and functioning, it is generally assumed that ribosomal-protein genes are unlikely to undergo horizontal transfer. However, with the accumulation of numerous complete genome sequences of prokaryotes, several paralogous pairs of ribosomal protein genes have been identified. Here we analyze all such cases and attempt to reconstruct the evolutionary history of these ribosomal proteins.

Results

Complete bacterial genomes were searched for duplications of ribosomal proteins. Ribosomal proteins L36, L33, L31, S14 are each duplicated in several bacterial genomes and ribosomal proteins L11, L28, L7/L12, S1, S15, S18 are so far duplicated in only one genome each. Sequence analysis of the four ribosomal proteins, for which paralogs were detected in several genomes, two of the ribosomal proteins duplicated in one genome (L28 and S18), and the ribosomal protein L32 showed that each of them comes in two distinct versions. One form contains a predicted metal-binding Zn-ribbon that consists of four conserved cysteines (in some cases replaced by histidines), whereas, in the second form, these metal-chelating residues are completely or partially replaced. Typically, genomes containing paralogous genes for these ribosomal proteins encode both versions, designated C+ and C-, respectively. Analysis of phylogenetic trees for these seven ribosomal proteins, combined with comparison of genomic contexts for the respective genes, indicates that in most, if not all cases, their evolution involved a duplication of the ancestral C+ form early in bacterial evolution, with subsequent alternative loss of the C+ and C- forms in different lineages. Additionally, evidence was obtained for a role of horizontal gene transfer in the evolution of these ribosomal proteins, with multiple cases of gene displacement 'in situ', that is, without a change of the gene order in the recipient genome.

Conclusions

A more complex picture of evolution of bacterial ribosomal proteins than previously suspected is emerging from these results, with major contributions of lineage-specific gene loss and horizontal gene transfer. The recurrent theme of emergence and disruption of Zn-ribbons in bacterial ribosomal proteins awaits a functional interpretation.     

Primary structures of five ribosomal proteins from the archaebacterium Halobacterium marismortui and their structural relationships to eubacterial and eukaryotic ribosomal proteins

The complete amino acid sequences of ribosomal proteins L9, L20, L21/22, L24 and L32 from the archaebacterium Halobacterium marismortui were determined. The comparison of the sequences of these proteins with those from other organisms revealed that proteins L21/22 and L24 are homologous to ribosomal protein Yrp29 from yeast and L19 from rat, respectively, and that H. marismortui L20 is homologous to L30 from eubacteria. H. marismortui ribosomal protein L9 showed sequence homology to both L29 from yeast and L15 from eubacteria. No homologous protein was found for H. marismortui L32. These results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes.