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.     

Amino acid sequences of ribosomal proteins S11 from Bacillus stearothermophilus and S19 from Halobacterium marismortui Comparison of the ribosomal protein S11 family

The complete amino acid sequences of ribosomal proteins S11 from the Gram-positive eubacterium Bacillus stearothermophilus and of S19 from the archaebacterium Halobacterium marismortui have been determined. A search for homologous sequences of these proteins revealed that they belong to the ribosomal protein S11 family. Homologous proteins have previously been sequenced from Escherichia coli as well as from chloroplast, yeast and mammalian ribosomes. A pairwise comparison of the amino acid sequences showed that Bacillus protein S11 shares 68% identical residues with S11 from Escherichia coli and a slightly lower homology (52%) with the homologous chloroplast protein. The halophilic protein S19 is more related to the eukaryotic (45–49%) than to the eubacterial counterparts (35%)     

Primary structures of ribosomal proteins from the archaebacterium Halobacterium marismortui and the eubacterium Bacillus stearothermophilus.

Approximately 40 ribosomal proteins from each Halobacterium marismortui and Bacillus stearothermophilus have been sequenced either by direct protein sequence analysis or by DNA sequence analysis of the appropriate genes. The comparison of the amino acid sequences from the archaebacterium H marismortui with the available ribosomal proteins from the eubacterial and eukaryotic kingdoms revealed four different groups of proteins: 24 proteins are related to both eubacterial as well as eukaryotic proteins. Eleven proteins are exclusively related to eukaryotic counterparts. For three proteins only eubacterial relatives-and for another three proteins no counterpart-could be found. The similarities of the halobacterial ribosomal proteins are in general somewhat higher to their eukaryotic than to their eubacterial counterparts. The comparison of B stearothermophilus proteins with their E coli homologues showed that the proteins evolved at different rates. Some proteins are highly conserved with 64-76% identity, others are poorly conserved with only 25-34% identical amino acid residues.     

Zinc finger-like motifs in rat ribosomal proteins S27 and S29.

The primary structures of the rat 40S ribosomal subunit proteins S27 and S29 were deduced from the sequences of nucleotides in recombinant cDNAs and confirmed by determination of amino acid sequences in the proteins. Ribosomal protein S27 has 83 amino acids and the molecular weight is 9,339. Hybridization of cDNA to digests of nuclear DNA suggests that there are 4-6 copies of the S27 gene; the mRNA for the protein is about 620 nucleotides in length. Ribosomal protein S29 has 55 amino acids and the molecular weight is 6,541. There are 14-17 copies of the S29 gene and its mRNA is about 500 nucleotides in length. Rat ribosomal protein S29 is related to several members of the archaebacterial and eubacterial S14 family of ribosomal proteins. S27 and S29 have zinc finger-like motifs as do other proteins from eukaryotic, archaebacterial, eubacterial, and mitochondrial ribosomes. Moreover, ribosomes and ribosomal subunits appear to contain zinc and iron as well.     

A small basic ribosomal protein in Sulfolobus solfataricus equivalent to L46 in yeast: Structure of the protein and its gene

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene (L46e) has been confirmed by partial amino acid sequencing. The protein shows substantial sequence homology to the eukaryotic ribosomal proteins L39 in rat and L46 in yeast. There is no sequence homology to any of the eubacterial ribosomal proteins suggesting that this protein is absent in the eubacterial ribosome.     

Primary structure of the archaebacterial Methanococcus vannielii ribosomal protein L12. Amino acid sequence determination, oligonucleotide hybridization, and sequencing of the gene

The primary structure of ribosomal protein L12 from Methanococcus vannielii has been determined by direct amino acid sequence analysis with automated liquid phase Edman degradation of the entire protein and manual 4-N,N'-dimethylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate sequencing of fragments obtained by enzymatic digestion and by partial acid hydrolysis. The knowledge of the amino acid sequences of these various fragments allowed the synthesis of two oligonucleotide probes complementary to the 5'- and the 3'-end of the gene, and they were used for hybridization with digested M. vannielii chromosomal DNA. Both oligonucleotide probes gave similar and clear hybridization signals. The plasmid pMvaX1 containing the entire gene of protein L12 was obtained. The nucleotide sequence complemented the partial amino acid sequence, and it is in full agreement with the protein sequence and the amino acid analysis. Comparison of secondary structural elements and hydrophobicity plots of the M. vannielii protein L12 with the known L12 sequences derived from other archaebacterial and eukaryotic sources show strong homologies among these sequences. They contain an exceptional highly conserved hydrophilic sequence area in the C-terminal part of the proteins. In comparison with eubacterial L12 proteins, the conservation is reduced to single amino acid residues. However, the eubacterial L12 proteins have hydrophilic regions similar to those of L12 from M. vannielii. These regions are predicted to be located at the surface of the proteins, as has been proven to be the case in crystallized Escherichia coli L12 protein. It is possible that the strongly conserved hydrophilic sequence regions form part of the factor-binding domain.     

A small basic ribosomal protein in Sulfolobus solfataricus equivalent to L46 in yeast: structure of the protein and its gene

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene (L46e) has been confirmed by partial amino acid sequencing. The protein shows substantial sequence homology to the eukaryotic ribosomal proteins L39 in rat and L46 in yeast. There is no sequence homology to any of the eubacterial ribosomal proteins suggesting that this protein is absent in the eubacterial ribosome.     

Structural motifs of the bacterial ribosomal proteins S20, S18 and S16 that contact rRNA present in the eukaryotic ribosomal proteins S25, S26 and S27A,respectively

The majority of constitutive proteins in the bacterial 30S ribosomal subunit have orthologues in Eukarya and Archaea. The eukaryotic counterparts for the remainder (S6, S16, S18 and S20) have not been identified. We assumed that amino acid residues in the ribosomal proteins that contact rRNA are to be constrained in evolution and that the most highly conserved of them are those residues that are involved in forming the secondary protein structure. We aligned the sequences of the bacterial ribosomal proteins from the S20p, S18p and S16p families, which make multiple contacts with rRNA in the Thermus thermophilus 30S ribosomal subunit (in contrast to the S6p family), with the sequences of the unassigned eukaryotic small ribosomal subunit protein families. This made it possible to reveal that the conserved structural motifs of S20p, S18p and S16p that contact rRNA in the bacterial ribosome are present in the ribosomal proteins S25e, S26e and S27Ae, respectively. We suggest that ribosomal protein families S20p, S18p and S16p are homologous to the families S25e, S26e and S27Ae, respectively.     

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.     

Molecular phylogenies based on ribosomal protein L11, L1, L10, and L12 sequences

Summary Available sequences that correspond to the E. coli ribosomal proteins L11, L1, L10, and L12 from eubacteria, archaebacteria, and eukaryotes have been aligned. The alignments were analyzed qualitatively for shared structural features and for conservation of deletions or insertions. The alignments were further subjected to quantitative phylogenetic analysis, and the amino acid identity between selected pairs of sequences was calculated. In general, eubacteria, archaebacteria, and eukaryotes each form coherent and well-resolved nonoverlapping phylogenetic domains. The degree of diversity of the four proteins between the three groups is not uniform. For L11, the eubacterial and archaebacterial proteins are very similar whereas the eukaryotic L11 is clearly less similar. In contrast, in the case of the L12 proteins and to a lesser extent the L10 proteins, the archaebacterial and eukaryotic proteins are similar whereas the eubacterial proteins are different. The eukaryotic L1 equivalent protein has yet to be identified. If the root of the universal tree is near or within the eubacterial domain, our ribosomal protein-based phylogenies indicate that archaebacteria are monophyletic. The eukaryotic lineage appears to originate either near or within the archaebacterial domain. Correspondence to: P. Dennis     

Evolutionary divergence and salinity-mediated selection in halophilic archaea.

Halophilic (literally salt-loving) archaea are a highly evolved group of organisms that are uniquely able to survive in and exploit hypersaline environments. In this review, we examine the potential interplay between fluctuations in environmental salinity and the primary sequence and tertiary structure of halophilic proteins. The proteins of halophilic archaea are highly adapted and magnificently engineered to function in an intracellular milieu that is in ionic balance with an external environment containing between 2 and 5 M inorganic salt. To understand the nature of halophilic adaptation and to visualize this interplay, the sequences of genes encoding the L11, L1, L10, and L12 proteins of the large ribosome subunit and Mn/Fe superoxide dismutase proteins from three genera of halophilic archaea have been aligned and analyzed for the presence of synonymous and nonsynonymous nucleotide substitutions. Compared to homologous eubacterial genes, these halophilic genes exhibit an inordinately high proportion of nonsynonymous nucleotide substitutions that result in amino acid replacement in the encoded proteins. More than one-third of the replacements involve acidic amino acid residues. We suggest that fluctuations in environmental salinity provide the driving force for fixation of the excessive number of nonsynonymous substitutions. Tinkering with the number, location, and arrangement of acidic and other amino acid residues influences the fitness (i.e., hydrophobicity, surface hydration, and structural stability) of the halophilic protein. Tinkering is also evident at halophilic protein positions monomorphic or polymorphic for serine; more than one-third of these positions use both the TCN and the AGY serine codons, indicating that there have been multiple nonsynonymous substitutions at these positions. Our model suggests that fluctuating environmental salinity prevents optimization of fitness for many halophilic proteins and helps to explain the unusual evolutionary divergence of their encoding genes.     

YmL9, a nucleus-encoded mitochondrial ribosomal protein of yeast, is homologous to L3 ribosomal proteins from all natural kingdoms and photosynthetic organelles.

The nuclear gene for mitochondrial ribosomal protein YmL9 (MRP-L9) of yeast has been cloned and sequenced. The deduced amino acid sequence characterizes YmL9 as a basic (net charge + 30) protein of 27.5 kDa with a putative signal peptide for mitochondrial import of 19 amino acid residues. The intact MRP-L9 gene is essential for mitochondrial function and is located on chromosome XV or VII. YmL9 shows significant sequence similarities to Escherichia coli ribosomal protein L3 and related proteins from various organisms of all three natural kingdoms as well as photosynthetic organelles (cyanelles). The observed structural conservation is located mostly in the C-terminal half and is independent of the intracellular location of the corresponding genes [Graack, H.-R., Grohmann, L. & Kitakawa, M. (1990) Biol. Chem. Hoppe Seyler 371, 787-788]. YmL9 shows the highest degree of sequence similarity to its eubacterial and cyanelle homologues and is less related to the archaebacterial or eukaryotic cytoplasmic ribosomal proteins. Due to their high sequence similarity to the YmL9 protein two mammalian cytoplasmic ribosomal proteins [MRL3 human and rat; Ou, J.-H., Yen, T. S. B., Wang, Y.-F., Kam, W. K. & Rutter, W. J. (1987) Nucleic Acids Res. 15, 8919-8934] are postulated to be true nucleus-encoded mitochondrial ribosomal proteins.     

Comparative studies of ribosomal proteins and their genes from Methanococcus vannielii and other organisms

Using data from a partial protein sequence analysis of ribosomal proteins derived from the archaebacterium Methanococcus vannielii, oligonucleotide probes were synthesized. The probes enabled us to localize several ribosomal protein genes and to determine their nucleotide sequences. The amino acid sequences that were deduced from the genes correspond to proteins L12 and L10 from the rif operon, according to the genome organization in Escherichia coli, and to proteins L23 and L2, which have comparable locations, as in the Escherichia coli S10 operon. Various degrees of similarity were found when the four proteins were compared with the corresponding ribosomal proteins of prokaryotic or eukaryotic organisms. The highest sequence homology was found in counterparts from other archaebacteria, such as Halobacterium marismortui, Halobacterium halobium, or Sulfolobus. In general, the M. vannielii protein sequences were more related to the eukaryotic kingdom than to the Gram-positive or Gram-negative eubacteria. On the other hand, the organization of the ribosomal protein genes clearly follows the operon structure of the Escherichia coli genome and is different from the monocistronic eukaryotic gene arrangements. The protein coding regions were not interrupted by introns. Furthermore, the Shine-Dalgarno type sequences of methanogenic bacteria are homologous with those of eubacteria, and also their terminator regions are similar.     

Extended N-terminal sequencing of proteins of the large ribosomal subunit from yeast mitochondria

We have determined the N-termini of 26 proteins of the large ribosomal subunit from yeast mitochondria by direct amino acid micro-sequencing. The N-terminal sequences of proteins YmL33 and YmL38 showed a significant similarity to eubacterial ribosomal (r-) proteins L30 and L14, respectively. In addition, several proteins could be assigned to their corresponding yeast nuclear genes. Based on a comparison of the protein sequences deduced from the corresponding DNA regions with the N-termini of the mature proteins, the putative leader peptides responsible for mitochondrial matrix-targeting were compiled. In most leader sequences a relative abundance of aromatic amino acids, preferentially phenylalanine, was found.     

Structure, function, and evolution of the family of superoxide dismutase proteins from halophilic archaebacteria.

The protein sequences of seven members of the superoxide dismutase (SOD) family from halophilic archaebacteria have been aligned and compared with each other and with the homologous Mn and Fe SOD sequences from eubacteria and the methanogenic archaebacterium Methanobacterium thermoautotrophicum. Of 199 common residues in the SOD proteins from halophilic archaebacteria, 125 are conserved in all seven sequences, and 64 of these are encoded by single unique triplets. The 74 remaining positions exhibit a high degree of variability, and for almost half of these, the encoding triplets are connected by at least two nonsynonymous nucleotide substitutions. The majority of nucleotide substitutions within the seven genes are nonsynonymous and result in amino acid replacement in the respective protein; silent third-codon-position (synonymous) substitutions are unexpectedly rare. Halophilic SODs contain 30 specific residues that are not found at the corresponding positions of the methanogenic or eubacterial SOD proteins. Seven of these are replacements of highly conserved amino acids in eubacterial SODs that are believed to play an important role in the three-dimensional structure of the protein. Residues implicated in formation of the active site, catalysis, and metal ion binding are conserved in all Mn and Fe SODs. Molecular phylogenies based on parsimony and neighbor-joining methods coherently group the halophile sequences but surprisingly fail to distinguish between the Mn SOD of Escherichia coli and the Fe SOD of M. thermoautotrophicum as the outgroup. These comparisons indicate that as a group, the SODs of halophilic archaebacteria have many unique and characteristic features. At the same time, the patterns of nucleotide substitution and amino acid replacement indicate that these genes and the proteins that they encode continue to be subject to strong and changing selection. This selection may be related to the presence of oxygen radicals and the inter- and intracellular composition and concentration of metal cations.     

The primary structure of rat ribosomal proteins: the amino acid sequences of L27a and L28 and corrections in the sequences of S4 and S12

The amino acid sequences of rat ribosomal proteins L27a and L28 were deduced from the sequences of nucleotides in recombinant cDNAs and confirmed from the NH2-terminal amino acid sequences of the proteins. L27a contains 147 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 16 476. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 18-22 copies of the L27a gene. The mRNA for the protein is about 600 nucleotides in length. L27a is homologous to mouse L27a (there are 3 amino acid changes) and to yeast L29. Rat ribosomal protein L28 has 136 amino acids (its NH2-terminal methionine is also processed after translation) and has a molecular weight of 15 707. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 9 or 10 copies of the L28 gene. The mRNA for the protein is about 640 nucleotides in length. L28 contains a possible internal duplication of 9 residues. Corrections are recorded in the sequences reported before for rat ribosomal proteins S4 and S12.     

A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein

A rice (Oryza sativa L.) cDNA clone coding for the cytoplasmic ribosomal protein L5, which associates with 5 S rRNA for ribosome assembly, was cloned and its nucleotide sequence was determined. The primary structure of rice L5, deduced from the nucleotide sequence, contains 294 amino acids and has intriguing features some of which are also conserved in other eucaryotic homologues. These include: four clusters of basic amino acids, one of which may serve as a nucleolar localization signal; three repeated amino acid sequences; the conservation of glycine residues. This protein was identified as the nuclear-encoded cytoplasmic ribosomal protein L5 of rice by sequence similarity to other eucaryotic ribosomal 5 S RNA-binding proteins of rat, chicken, Xenopus laevis, and Saccharomyces cerevisiae. Rice L5 shares 51 to 62% amino acid sequence identity with the homologues. A group of ribosomal proteins from archaebacteria including Methanococcus vanniellii L18 and Halobacterium cutirubrum L13, which are known to be associated with 5 S rRNA, also related to rice L5 and the other eucaryotic counterparts, suggesting an evolutionary relationship in these ribosomal 5 S RNA-binding proteins.     

The primary structure of rat ribosomal protein L3.

The amino acid sequence of the rat 60S ribosomal subunit protein L3 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L3 has 403 amino acids and has a molecular weight of 46,106. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7 to 9 copies of the L3 gene. The mRNA for the protein is about 1,400 nucleotides in length. Rat L3 is homologous to ribosomal proteins from other eukaryotes and to proteins from eubacterial, archaebacterial, and chloroplast ribosomes.