The amino acid sequence of two small ribosomal proteins from Bacillus stearothermophilus

The low-Mr proteins (tentatively called protein I and II) were purified from 2 M NaCl extracts of the Bacillus stearothermophilus ribosome. Their amino acid sequences have been determined from the peptides obtained by digestion with trypsin, chymotrypsin, and pepsin, and by cleavage with CNBr, using the micro-DABITC/PITC double-coupling method [FEBS Lett. (1978) 93, 205-214]. Protein I contains 56 residues and has an Mr of 6514. Protein II had 37 residues with an Mr of 4361. The amino acid sequence of protein I shows significant similarity to L32 from E. coli, whereas that of protein II is slightly, if at all, related to ribosomal protein L34 from E. coli.     

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.     

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.     

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.     

The N-terminal sequence of ribosomal protein L10 from the archaebacterium Halobacterium marismortui and its relationship to eubacterial protein L6 and other ribosomal proteins

The amino-terminal sequence of ribosomal protein L10 from Halobacterium marismortui has been determined up to residue 54, using both a liquid- and a gas-phase sequenator. The two sequences are in good agreement. The protein is clearly homologous to protein HcuL10 from the related strain Halobacterium cutirubrum. Furthermore, a weaker but distinct homology to ribosomal protein L6 from Escherichia coli and Bacillus stearothermophilus can be detected. In addition to 7 identical amino acids in the first 36 residues in all four sequences a number of conservative replacements occurs, of mainly hydrophobic amino acids. In this common region the pattern of conserved amino acids suggests the presence of a beta-alpha fold as it occurs in ribosomal proteins L12 and L30. Furthermore, several potential cases of homology to other ribosomal components of the three ur-kingdoms have been found.     

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%)     

Reassignment of residue 122 in the myoglobin from the killer whale,Orcinus orca

Summary The complete amino acid sequence of the major component myoglobin from killer whale,Orcinus orca, was determined by automated Edman degradation. In this study residue 122 was found to be glutamic acid instead of glutamine as was originally reported (Castillo et al. 1977). This reassignment affects the phylogenetic relationship of killer whale myoglobin with the myoglobins from other closely related cetacean species and also affects studies concerned with the physical parameters of the protein.This is the 114th paper in a series dealing with coordination complexes and catalytic properties of proteins and related substances. For the preceding paper see Neireiter et al. 1979. This work was supported by Public Health Service Research Grant HL-05556     

Phosphorylation of ribosomal proteins S3, L1 and L24 during spherulation in Physarum polycephalum

The phosphate content of ribosomal proteins S3, L1 and L24 has been determined in the course of spherulation of Physarum polycephalum. The major phosphoprotein, S3, was completely dephosphorylated after 4 h of differentiation. The phosphate content of L1 and L24 was not altered during the differentiation. The cellular level of ATP remained constant for at least 5 h. A 3-fold reduction of cyclic AMP concentration occurred in the first hour, followed by a slow increase to a final value of twice the level observed in growing cells. The results showed that the phosphorylation of ribosomal proteins is regulated by at least two different mechanisms and that the dephosphorylation of S3 is not induced by a lack of cellular ATP. Although cyclic AMP might trigger the dephosphorylation of S3, the phosphate content of this protein remained at a very low value even when the cellular concentration of cyclic AMP rose significantly. Since the polysome level remains constant during the first 24 h of spherulation, the phosphorylation of S3 is not necessary for active protein synthesis and the phosphorylation of L1 and L24 is not involved in ribosome inactivation, which occurs after 24 h.     

Genomic style of proteins: concepts, methods and analyses of ribosomal proteins from 16 microbial species

We have introduced the concept of genomic 'style' of proteins. By style we understand those properties of a large set of proteins which are specific to the genome of one species (species primary-self) and different from the genome of another species (species contrasted-self). To characterise the style, we took advantage of the frequencies of amino acids and dipeptides present in non-identical segments of the complete set of orthologous ribosomal proteins encoded by 16 microbial species. We confirm the dependence of the overall amino acid composition on the genomic (G+C) content, and introduce a rectification procedure making it possible to extricate appropriate species-specific characteristics, which are no longer related to this content. The rectified frequencies are used to calculate inter-species distance matrices, and to build genomic evolutionary trees. Remarkably, the phylograms derived from the frequencies of non-identical residues in proteins closely resemble the classical phylograms based upon the conservation of identical residues in ribosomal RNAs. We believe that the concept of genomic style of proteins can be a useful tool for the study of evolution.     

Nucleotide sequence of four genes encoding ribosomal proteins from the 'S10 and spectinomycin' operon equivalent region in the archaebacterium Halobacterium marismortui

Four genes encoding ribosomal proteins HmaS17, HmaL14, HmaL24 and HS3, have been identified in the lambda EMBL3 clone PP*7 from a genomic library of the archaebacterium Halobacterium marismortui. The clone contains genes from the 'S10 and spectinomycin' operon equivalent region. Three of the deduced proteins are homologous to the corresponding Escherichia coli and Methancoccus vannielii S17, L14 and L24 proteins, as well as to eukaryotic proteins from rat or yeast. HS3 was identified as an extra protein corresponding to the gene product for orfc in M. vannielii and the eukaryotic ribosomal protein RS4 from rat. The equivalence of HmaL24 (HL16) and E. coli L24, which share only 28% identical amino acid residues, could now be shown by localizing the HmaL24 gene at the same position in the cluster.     

Molecular cloning and nucleotide sequence of the gene for the ribosomal protein S11 from the archaebacterium Halobacterium marismortui

The gene encoding ribosomal protein S11 (Escherichia coli S15 homologue) from Halobacterium marismortui was cloned employing two synthetic oligonucleotide mixtures, 23 and 32 bases in length, as hybridization probes. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (1300 base pairs) were then determined by the dideoxy chain termination method. Comparison of the nucleotide sequence of the H. marismortui S11 gene with that of the E. coli S15 gene (rpsO) showed that the 3'-end of the S11 gene can be aligned with the entire E. coli S15 gene, sharing 44% identical nucleotides. It has been found that the S11 gene has a higher G + C content (G + C = 65%) than that of the E. coli S15 gene (G + C = 53%). This increase in G + C content specifically shows up as a preference for G + C in the 3rd position of the codon. Upstream of the S11 gene, an archaebacterial promoter sequence (GGACTTTCA) and a putative ribosomal binding site (GCGGT) have been found, 88 and 15 (or 24) base pairs from the initiation codon of the gene. In addition, an open reading frame could be identified immediately after the stop codon for the S11 gene. Northern blotting analysis using the S11 coding region as probe has shown that the S11 gene is located on a 2.4-kilobase mRNA, suggesting that it is cotranscribed with other downstream gene(s).     

The complete primary structure of ribosomal protein L1 fromThermus thermophilus

The primary structure of the 23S rRNA binding ribosomal protein L1 from the 50S ribosomal subunit ofThermus thermophilus ribosomes has been elucidated by direct protein sequencing of selected peptides prepared by enzymatic and chemical cleavage of the intact purified protein. The polypeptide chain contains 228 amino acids and has a calculated molecular mass of 24,694 D. A comparison with the primary structures of the corresponding proteins fromEscherichia coli andBacillus stearothermophilus reveals a sequence homology of 49% and 58%, respectively. With respect to both proteins, L1 fromT. thermophilus contains particularly less Ala, Lys, Gln, and Val, whereas its content of Glu, Gly, His, Ile, and Arg is higher. In addition, two fragments obtained by limited proteolysis of the intact, unmodified protein were characterized.     

Conservation and variation of ribosomal proteins in several species of the cellular slime molds Dictyostelium and Polysphondylium

Genus- and species-specific composition of ribosomal proteins was investigated in four species of the genus Dictyostelium (D. discoideum, D. purpureum, D. murcoroides and D. giganteum) and two species of the genus Polysphondylium (P. pallidum and P. violaceum). Ribosomal proteins were resolved by a high-resolution, two-dimensional gel method. In general, the numbers and distributions for the majority of ribosomal proteins were similar within the species of each genus, although some differences were detected. More differences were observed between Dictyostelium and Polysphondylium than among the individual species within each genus. Stage-specific ribosomal proteins previously demonstrated in D. discoideum were found to be developmentally regulated in other Dictyostelium species, and in both Polysphondylium species. The study shows that ribosomal proteins may be a potentially useful new biochemical parameter for the molecular taxonomy of the cellular slime molds.     

Purification and characterization of ribosomal proteins from the 30 S subunit of the extreme halophile Halobacterium marismortui

Ribosomal proteins were extracted from 30 S subunits of Halobacterium marismortui under native conditions.Their separation was based on gel filtration and hydrophobic chromatography, performed at a concentration of 3.2 M KC1 to avoid denaturation. A total of nine proteins were isolated, purified and identified by partial amino-terminal sequences and two-dimension a gel electrophoresis. There is a high degree of sequence homology with 30 S proteins from H. cutirubrum, and also some with 30 (S) proteins of eubacteria.Proton NMR data indicate unfolding of the proteins in low salt. One of the proteins, however, retains its secondary structure at a salt concentration as low as 0.1 M NaCl, and even in 8 M urea. One reason for this outstanding stability could be the high proportion (50%) of β-structure in this protein as determined from circular dichroism measurements. In general, there is a higher β-sheet content than for 30 S proteins from Escherichia coli. Measurements of Stokes radii indicate several of the proteins to have a rather elongated shape. One of these is a complex consisting of L3/L4 and L20, similar to the LI-complex from E. co&.The presence of this 50 S complex in the preparation of the small subunit suggests a location on the interface between the subunits.     

Organization and nucleotide sequence of ten ribosomal protein genes from the region equivalent to the spectinomycin operon in the archaebacterium Halobacterium marismortui.

Summary We have created missense mutations in the indirect flight muscle (IFM)-specific Act88F actin gene of Drosophila melanogaster by random in vitro mutagenesis. Following P element-mediated transformation into wild-type flies and subsequent transfer of the inserts into Act88F null strains, the effects of the actin mutants on the structure and function of the IFMs were examined. All of the mutants were antimorphic for flight ability. E316K and G368E formed muscle with only relatively small defects in structure whilst the others produced IFMs with large amounts of disruption. E334K formed filaments but lacked Z discs. V339I formed no muscle structure in null flies and did not accumulate actin. E364K and G366D both had relatively stable actin but did not form myofibrils. Using an in vitro polymerisation assay we found no significant effects on the ability of the mutant actins to polymerise. E364K and G366D also caused a strong induction of heat shock protein (hsp) synthesis at normal temperatures and accumulated large amounts of hsp22 which, together with the mutant actin, was resistant to detergent extraction. Both E316K and E334K caused a weak induction of hsp synthesis. We discuss how the stability, structure and function of the different mutant actins affects myofibril assembly and function, and the induction of hsps.     

Amino acid sequence of the ribosomal protein HS23 from the halophilicHaloarcula marismortui and homology studies to other ribosomal proteins

The ribosomal protein HS23 from the 30S subunit of the extreme halophilicHaloarcula marismortui, belonging to the group of archaea, was isolated either by RP-HLPLC or two-dimensional polyacrylamide gel electrophoresis. The complete amino acid sequence was determined by automated N-terminal microsequencing. The protein consists of 123 residues with a corresponding molecular mass of 12,552 Da as determined by electrospray mass spectroscopy; the pI is 11.04. Homology studies reveal similarities to the eukaryotic ribosomal protein S8 fromHomo sapiens, Rattus norvegicus, Leishmania major, andSaccharomyces cerevisiae.Abbreviations H. marismortui Haloarcula marismortui - PVDF polyvinylidene difluoride - PTH phenylthiohydantoin - RP-HPLC reversed-phase high-performance liquid chromatography - TFA trifluoro acetic acid - TP30 total protein mixture from the 30S ribosomal subunit ofH. marismortui