Particle weights and protein composition of the ribosomal subunits of the extremely thermoacidophilic archaebacterium Caldariella acidophila.

1. The ribosomal subunits of one thermoacidophilic archaebacterium (Caldariella acidophila) and of two reference eubacterial species (Bacillus acidocaldarius, Escherichia coli) were compared with respect to ribosome mass and protein composition by (i) equilibrium-density sedimentation of the particles in CsCl and (ii) gel-electrophoretic estimations of the molecular weights of the protein and the rRNA. 2. By either procedure, it is estimated that synthetically active archaebacterial 30S subunits (52% protein by wt.) are appreciably richer in protein than the corresponding eubacterial particles (31% protein by wt.) 3. The greater protein content of the archaebacterial 30S subunits is accounted for by both a larger number and a greater average molecular weight of the subunit proteins; specifically, C. acidophila 30S subunits yield 28 proteins whose combined mass is 0.6 X 10(6) Da, compared with 20 proteins totalling 0.35 X 10(6) Da mass for eubacterial 30S subunits. 4. No differences in protein number are detected among the large subunits, but C. acidophila 50S subunits exhibit a greater number-average molecular weight of their protein components than do eubacterial 50S particles. 5. Particle weights estimated by either buoyant-density data, or molecular weights of rRNA plus protein, agree to within less than 2%. By either procedure C. acidophila 30S subunits 1.15 X 10(6) Da mass) are estimated to be about 300 000 Da heavier than their eubacterial counterparts (0.87 X 10(6) Da mass); a smaller difference. 0.15 X 10(6) Da, exists between the archaebacterial and the eubacterial 50S subunits (respectively 1.8 X 10(6) and 1.65 X 10(6) Da). It is concluded that the heavier-than-eubacterial mass of the C. acidophila ribosomes resides principally in their smaller subunits.     

A region in the C-terminal domain of ribosomal protein SA required for binding of SA to the human 40S ribosomal subunit

The human ribosomal protein SA, known also as a precursor of the cell-surface laminin receptor, LAMR, is a protein of the 40S ribosomal subunit. It is homologous to eubacterial ribosomal protein S2p, but has a eukaryote-specific C-terminal domain (CTD) that is responsible in LAMR for the binding of laminin as well as prions and several viruses. Using serial deletions in the SA CTD, we showed that region between amino acids 236-262 is required for binding of the protein to 40S ribosomal subunits. All SA mutants containing this region protected nucleotides in hairpin 40 (which is not bound to any protein in the eubacterial 30S ribosomal subunit) of the 18S rRNA from hydroxyl radical attack. Comparison of our data with the cryo-EM models of the mammalian 40S ribosomal subunit allowed us to locate the SA CTD in the spatial structure of the 40S subunit.     

The ribosomal genes of Mycoplasma capricolum

The nucleotide sequence of 5S rRNA from Mycoplasma capricolum is more similar to that of the gram-positive bacteria than that of the gram-negative bacteria. The presence of two copies of rRNA genes in M. capricolum genome has been demonstrated. The two different rRNA gene clusters have been cloned in E. coli plasmid vectors and analyzed for the rRNA gene organizations, demonstrating that the gene arrangement is in the order of 16S, 23S, and 5S rDNA. The ribosomes of M. capricolum contain about 30 species of proteins in 50S and 20 in 30S subunits. The number and size of the ribosomal proteins are not significantly different from those of other eubacterial ribosomes.     

Structural analysis of 5S rRNA, 5S rRNA-protein complexes and ribosomes employing RNase H and d(GTTCGG)

The hybridization of d(GTTCGG) to eubacterial 5S rRNAs, 5S rRNA-protein complexes, 70S ribosomes and 50S and 30S ribosomal subunits was investigated. This oligonucleotide, which may be considered to be an analogue of the T psi CG loop of tRNAs, was chosen in order to investigate a possible interaction between tRNAs with ribosomal components during protein synthesis. The hybridization was analysed by RNase H hydrolysis studies and, in the case of the ribosomes and ribosomal subunits, in addition with the radioactively labelled oligodeoxyribonucleotide in binding studies. The results obtained lead to the conclusion that nucleotides in loop c, i.e. positions 42-47, are available for oligonucleotide interaction in free Escherichia coli and Bacillus stearothermophilus 5S rRNAs and not available in the corresponding 5S rRNA-protein complexes. The 70S ribosomes and ribosomal subunits did not interact with the oligonucleotide. Under the assumption that d(GTTCGG) is an analogue of the T psi CG loop of tRNAs and in view of the results obtained, we conclude that in the unprogrammed ribosomes the T psi CG loop of tRNAs does not interact via standard Watson-Crick base pairs with the ribosomal 5S, 16S or 23S RNAs.     

Polypeptide synthesis on ribosomes of an extreme thermophilic hydrogen bacterium Calderobacterium hydrogenophilum

Ribosomes of the extreme thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum interact with a broad spectrum of polyamines. In the absence of polyamines and at 70°C close to the growth optimum (75°C), high salt washed ribosomes lost their activity in the poly(UG)-directed polypeptide synthesis. At 70°C the in vitro system synthesized the polypeptide in the presence of spermidine, spermine or natural polyamines (tri-pentaamines) isolated from ribosomal extracts but not in the presence of putrescine. The activity of ribosomes was affected by a number of antibiotics interfering with functions of typical eubacterial 70S, such as tetracyclines, lincomycin, chloramphenicol and erythromycin. However, the ribosomes were relatively resistant to streptomycin and insensitive to 80S inhibitors, such as ricin and cycloheximide. The 30S and 50S subunits have structural features typical of eubacterial ribosomes.The authors dedicated this paper to Professor Ivan Málek on the occasion of his 80th birthdy to remember his contribution to the Czechoslovak microbiology     

In vitro incorporation of eubacterial, archaebacterial and eukaryotic 5S rRNAs into large ribosomal subunits of Bacillus stearothermophilus.

Bacillus stearothermophilus large ribosomal subunits were reconstituted in the presence of 5S rRNAs from different origins and tested for their biological activities. The results obtained have shown that eubacterial and archaebacterial 5S rRNAs can easily substitute for B. stearothermophilus 5S rRNA in the reconstitution, while eukaryotic 5S rRNAs yield ribosomal subunits with reduced biological activities. From our results we propose an interaction between nucleotides 42-47 of 5S rRNA and nucleotides 2603-2608 of 23S rRNA during the assembly of the 50S ribosomal subunit. Other experiments with eukaryotic 5.8S rRNAs reveal, if at all, a very low incorporation of these RNA species into the reconstituted ribosomes.     

Reconstitution of 50 S ribosomal subunits from Bacillus stearothermophilus with 5 S RNA from spinach chloroplasts and low-Mr RNA from mitochondria of Locusta migratoria and bovine liver

Reconstitution experiments with 50 S ribosomal subunits from Bacillus stearothermophilus demonstrate that spinach chloroplast 5 S rRNA can be incorporated into the bacterial ribosome and yield biologically active particles, thereby establishing the eubacterial nature of chloroplast 5 S rRNA. In contrast, mitochondria from Locusta migratoria or bovine liver do not appear to contain discrete, low-Mr RNAs, which can replace 5 S rRNA in the functional reconstitution of B. stearothermophilus ribosomes.     

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.     

Relatedness of archaebacterial RNA polymerase core subunits to their eubacterial and eukaryotic equivalents.

The sequence of the genes encoding the four largest subunits of the RNA polymerase of the archaebacterium Methanobacterium thermoautotrophicum was determined and putative translation signals were identified. The genes are more strongly homologous to eukaryotic than to eubacterial RNA polymerase genes. Analysis of the polypeptide sequences revealed colinearity of two pairs of adjacent archaebacterial genes encoding the B" and B' or A and C genes, respectively, with two eubacterial and two eukaryotic genes each encoding the two largest RNA polymerase subunits. This difference in sequence organization is discussed in terms of gene fusion in the course of evolution. The degree of conservation is much higher between the archaebacterial and the eukaryotic polypeptides than between the archaebacterial and the eubacterial enzyme. Putative functional domains were identified in two of the subunits of the archaebacterial enzyme.     

Structural and functional exchangeability of 5 S RNA species from the eubacterium E.coli and the thermoacidophilic archaebacterium Sulfolobus solfataricus.

The role of 5 S RNA within the large ribosomal subunit of the extremely thermophilic archaebacterium Sulfolobus solfataricus has been analysed by means of in vitro reconstitution procedures. It is shown that Sulfolobus 50 S subunits reconstituted in the absence of 5 S RNA are inactive in protein synthesis and lack 2-3 ribosomal proteins. Furthermore, it has been determined that in the course of the in vitro assembly process Sulfolobus 5 S RNA can be replaced by the correspondent RNA species of E.coli; Sulfolobus reconstituted particles containing the eubacterial 5 S molecule are stable and active in polypeptide synthesis at high temperatures.     

Determination of peptide regions on the surface of the eubacterial and archaebacterial ribosome by limited proteolytic digestion.

Limited proteolysis was used in combination with two-dimensional gel electrophoresis, blotting, and amino acid sequence analysis to investigate the surface of intact ribosomal subunits at the peptide and amino acid level. Surface sites of 14 ribosomal proteins from Escherichia coli 50S subunits were determined using proteases with different specificities. To assess the evolutionary conservation of ribosomal topography among eubacteria, large subunits from Bacillus stearothermophilus were also subjected to limited proteolysis. The results obtained indicate a conservation of the three-dimensional ribosomal structure at the peptide level. The data for the eubacterial ribosomes are in full agreement with the model of the 50S protein topography derived from immunological data. Furthermore, peptide surface regions of archaebacterial ribosomes have been investigated. The results presented in this work prove that limited proteolysis can successfully be applied to halophilic and thermophilic ribosomes from archaebacteria.     

The structure of the archaebacterial ribosomal protein S7 and its possible interaction with 16S rRNA.

Ribosomal protein S7 is one of the ubiquitous components of the small subunit of the ribosome. It is a 16S rRNA-binding protein positioned close to the exit of the tRNA, and it plays a role in initiating assembly of the head of the 30S subunit. Previous structural analyses of eubacterial S7 have shown that it has a stable alpha-helix core and a flexible beta-arm. Unlike these eubacterial proteins, archaebacterial or eukaryotic S7 has an N-terminal extension of approximately 60 residues. The crystal structure of S7 from archaebacterium Pyrococcus horikoshii (PhoS7) has been determined at 2.1 A resolution. The final model of PhoS7 consists of six major alpha-helices, a short 3(10)-helix and two beta-stands. The major part (residues 18-45) of the N-terminal extension of PhoS7 reinforces the alpha-helical core by well-extended hydrophobic interactions, while the other part (residues 46-63) is not visible in the crystal and is possibly fixed only by interacting with 16S rRNA. These differences in the N-terminal extension as well as in the insertion (between alpha1 and alpha2) of the archaebacterial S7 structure from eubacterial S7 are such that they do not necessitate a major change in the structure of the currently available eubacterial 16S rRNA. Some of the inserted chains might pass through gaps formed by helices of the 16S rRNA.     

Three-dimensional structure of the mammalian cytoplasmic ribosome

A three-dimensional reconstruction of the 80 S ribosome from rabbit reticulocytes has been calculated from low-dose electron micrographs of a negatively stained single-particle specimen. At 37 A resolution, the precise orientations of the 40 S and 60 S subunits within the monosome can be discerned. The translational domain centered on the upper portion of the subunit/subunit interface is quite open, allowing considerable space between the subunits for interactions with the non-ribosomal macromolecules involved in protein synthesis. Further, the cytosolic side of the monosome is strikingly more open than the membrane-attachment side, suggesting a greater ease of communication with the cytoplasm, which would facilitate the inwards and outwards diffusion of a number of ligands. Although the 60 S subunit portion of the 80 S structure shows essentially all of the major morphological features identified for the eubacterial 50 S large subunit, it appears to possess a region of additional mass that evidently accounts for the more ellipsoidal form of the eukaryotic subunit.     

Immunoprecipitation of 70S, 50S and 30S ribosomes ofEscherichia coli

Antibodies were raised in rabbits against 70S ribosomes, 50S and 30S ribosomal subunits individually. Purified immunoglobulins from the antiserum against each of the above ribosomal entities were tested for their capabilities of precipitating 70S, 50S and 30S ribosomes. The observations revealed the following: (i) The antiserum (IgG) raised against 70S ribosomes precipitates 70S ribosomes completely, while partial precipitation is seen with the subunits, the extent of precipitation being more with the 50S subunits than with 30S subunits; addition of 50S subunits to the 30S subunits facilitates the precipitation of 30S subunits by the antibody against 70S ribosomes. (ii) Antiserum against 50S subunits has the ability to immunoprecipitate both 50S and 70S ribosomes to an equal extent. (iii) Antiserum against 30S subunits also has the property of precipitating both 30S and 70S ribosomes. The differences in the structural organisation of the two subunits may account for the differences in their immunoprecipitability.     

Comparative Analysis of Eubacterial DNA Polymerase III Alpha Subunits

DNA polymerase III is one of the five eubacterial DNA polymerases that is re-sponsible for the replication of DNA duplex. Among the ten subunits of the DNApolymerase III core enzyme, the alpha subunit catalyzes the reaction for polymer-izing both DNA strands. In this study, we extracted genomic sequences of thealpha subunit from 159 sequenced eubacterial genomes, and carried out sequence-based phylogenetic and structural analyses. We found that all eubacterial genomeshave one or more alpha subunits, which form either homodimers or heterodimers.Phylogenetic and domain structural analyses as well as copy number variations ofthe alpha subunit in each bacterium indicate the classification of alpha subunit intofour basic groups: polC, dnaE1, dnaE2, and dnaE3. This classification is of essencein genome composition analysis. We also consolidated the naming convention toavoid further confusion in gene annotations.     

The structure and evolution of archaebacterial ribosomal RNAs

A cladistic analysis of 553 5S rRNA sequences has revealed a Ur-5S rRNA, the ancestor of all present-day 5S rRNA molecules. Previously stated characteristic differences between the eubacterial and eukaryotic molecules, namely, the length base-pairing schemes of helices D, can be used as a marker for the various archaebacterial branches. One model comprises Thermococcus, Thermoplasma, methanobacteria, and halobacteria; a second comprises the Sulfolobales; and a third is represented only by the single organism Octopus Spring species 1. A relaxed selection pressure on helix E with subsequent deletions is observed in Methanobacteriales, Methanococcales, and eubacteria. The secondary structures are supported by enzymatic digestion and chemical modification studies of the 5S rRNAs. Reconstitution of eubacterial 50S ribosomal subunits with 5S rRNA from Halobacterium and Thermoplasma has revealed 100% incorporation, while eukaryotic 5S rRNAs yielded a 50% incorporation. Relevant positions of the small-subunit rRNA are selected to answer the question of the monophyly of archaebacteria. Eight positions account for monophyly, eight for an ancestry of eubacteria with halophile methanogens and eukaryotes with eocytes (paraphyly of archaebacteria), and two for an ancestry of eubacteria with eocytes. A refinement of the neighborliness method of S. Sattath and A. Tversky resulted in a monophyly of archaebacteria when all positions are treated equally and in a paraphyly when tranversions are weighted twice over transitions.     

Inactivation of the 20S proteasome in Mycobacterium smegmatis

The 20S proteasome is an essential component of the cytosolic protein turnover apparatus of eukaryotic cells. In higher eukaryotes, the 20S proteasome is responsible for most cytosolic protein turnover and also generates peptides for subsequent presentation by the MHC class I pathway. Structurally, the eukaryotic 20S proteasome is extremely complex, being composed of 14 different subunits. Proteasomes with simplified subunit composition have been identified in certain eubacteria and archaebacteria but, in each case, the proteasome-containing organism is recalcitrant to further molecular genetic analyses. As a result, no in vivo characterization of a simplified eubacterial or archaebacterial proteasome has been reported. We have shown that the genetically tractable eubacterium Mycobacterium smegmatis contains a 20S proteasome, allowing the first in vivo characterization of a simplified 20S proteasome. We use a positive/negative selection scheme to inactivate the genes encoding 20S proteasome subunits and demonstrate that, in contrast to eukaryotic cells, M. smegmatis cells lacking intact proteasome genes are viable and phenotypically indistinguishable from congenic strains containing proteasomes. Implications for the evolution of the protein turnover apparatus are discussed.     

The structure of the 5 S ribosomal RNA of a member of the phylum of green non-sulfur bacteria and relatives

The 5 S rRNA sequence was determined for the bacterium Herpetosiphon strain Senghas Wie 2. It is the first 5 S RNA sequence reported for a member of the eubacterial phylum defined by green non-sulfur bacteria. The sequence fits into a consensus secondary structure model for eubacterial 5 S RNA. At four positions, the sequence shows substitutions with respect to strongly conserved nucleotides found in other hitherto examined eubacterial 5 S RNAs.