Mescaline-induced changes of brain-cortex ribosomes. Role of spermidine in counteracting the destabilizing effect of mescaline on brain-cortex ribosomes

1. The effect of spermidine on the mescaline-induced changes of brain-cortex ribosomes was studied by adding spermidine during the treatment of goat brain-cortex slices with mescaline. 2. Mescaline treatment of brain-cortex slices removed a portion of the endogenous spermidine from ribosomes and this removal was significantly prevented when spermidine was present during mescaline treatment. 3. Spermidine present during mescaline treatment of brain-cortex slices counteracted, to some extent, the destabilizing effect of mescaline on ribosomes with respect to heat denaturation. 4. Mescaline treatment of brain-cortex slices made ribosomes more susceptible to breakdown, releasing protein and RNA, and resulting in loss of ribosomal enzymic activities. However, spermidine present during mescaline treatment counteracted moderately the mescaline-induced ribosomal susceptibility to breakdown and ribosomal loss of enzymic activities. 5. Ribosomes of mescaline-treated cortex slices were rapidly degraded by ribonuclease and trypsin. However, if spermidine was present during mescaline treatment of brain-cortex slices the rates of degradation diminished.     

Mescaline-induced changes of brain-cortex ribosomes. Effect of mescaline on the hydrogen-bonded structure of ribonucleic acid of brain-cortex ribosomes

1. The action of mescaline sulphate on the hydrogen-bonded structure of the RNA constituent of ribosomes of goat brain-cortex slices was studied by using the hyperchromic effect of heating and formaldehyde reaction. 2. The ribosomal total RNA species of the mescaline-treated brain-cortex slices have a smaller proportion of hydrogen-bonded structure than the ribosomal RNA species of the untreated brain-cortex slices. 3. Mescaline also appears to have affected this lowering of hydrogen-bonded structure of the ribosomal 28S RNA of brain-cortex tissue.     

Effect of polyamines on the stability of brain-cortex ribosomes

1. Ribosomes isolated from the cortex tissue of goat brain contain very small amounts of spermidine and spermine. Ribosomes isolated from spermidine-treated slices have a higher spermidine content. 2. The polyamines partially prevent the temperature-dependent breakdown of ribosomes into acid-soluble nucleotides. 3. The ;melting' temperature of ribosomes rises slightly when the ribosomes are heated slowly in the presence of polyamines. 4. The pH-dependent breakdown of ribosomes into protein, RNA and acid-soluble nucleotide is markedly decreased by polyamines present in media in which ribosomes are suspended. 5. The breakdown of ribosomes in the presence of high concentrations of salts and EDTA is partially checked by the concurrent presence of polyamines. 6. Spermidine and spermine make ribosomes less susceptible to enzymic digestion by crystalline trypsin and ribonuclease.     

A comparative study of ribonuclease hydrolysis of rat brain-cortex and liver membrane-bound ribosomes

Because it has been proposed that the ribosome–membrane interaction is different in endoplasmic reticulum derived from a non-secretory and secretory cell we undertook a study to determine whether attachment of the ribosome to the membrane involved ribosomal RNA and if the rRNA in ribosomes derived from the two classes of cell possessed an altered susceptibility to RNAase (ribonuclease) hydrolysis. We found that brain ribosomes appeared to possess more regions accessible to nuclease attack, independent of whether a sequence-dependent RNAase (T₁) or a sterically hindered RNAase bound to Enzite polymer was employed. These results were independent of whether the ribosomes were membrane-bound or detached from the endoplasmic reticulum membranes, but at high RNAase concentration these differences became negligible. No conclusions, however, could be drawn as to whether ribosomal RNA is involved in the attachment of the ribosome to the endoplasmic reticulum membrane, because of the presence of endogeneous membrane-associated RNAases. Analysis of the rRNA fragments by polyacrylamide-gel electrophoresis suggests that the sites available for attack by low concentrations of nuclease in bound-ribosomes derived from brain cortex are different from those of liver.     

Isolation and characterization of membrane-bound ribosomes from nerve cell bodies of immature rat brain-cortex.

Free and membrane-bound ribosomes were isolated from neuronal perikarya of the immature rat brain-cortex. The two topographic forms of ribosomes were essentially free of contaminating organelles as shown by RNA, protein and marker enzyme analysis. Membrane-bound ribosomes amount to about a quarter of the total ribosomal population in neuronal perikarya. Both forms of ribosomes efficiently carried out cell-free protein synthesis but the membrane-bound fraction was more active than the free ribosomes.     

Structure and evolution of ribosomes

Ribosomes are the only cell organelles occurring in all organisms. E. coli ribosomes, which are the best characterized particles, consist of three RNAs and 53 proteins. All components have been isolated and characterized by chemical, physical and immunological methods. The primary structures of the RNAs and of all the proteins are known. Information about the secondary structure of the proteins derives from circular dichroism measurements and from secondary structure prediction methods. The tertiary structure is being studied by limited proteolysis, proton magnetic resonance and crystallization followed by X-ray analysis. Various methods are being used to elucidate the architecture of the ribosomal particle: three-dimensional image reconstruction of crystals of bacterial ribosomes and/or their subunits; immune electron microscopy; neutron scattering; protein-protein, protein-RNA and RNA-RNA crosslinking; total reconstitution of ribosomal subunits. The results from these studies yield valuable information on the architecture of the ribosomal particle. Many mutants have been isolated in which one or a few ribosomal proteins are altered or even deleted. The genetic and biochemical characterization of these mutants allows conclusions about the importance of these proteins for the function of the ribosome. Ribosomal proteins from various prokaryotic and eukaryotic species have been compared by two-dimensional gel electrophoresis, immunological methods, reconstitution and amino acid sequence analysis. These studies show a strong homology among prokaryotic ribosomal proteins but only a weak homology between proteins from prokaryotic and eukaryotic ribosomes. Comparison of the primary and secondary structures of the ribosomal RNAs from various organisms shows that the secondary structure of the RNA molecules has been strongly conserved throughout evolution.     

Immunochemical analysis of the structure of eukaryotic ribosomes

Summary Antibodies were prepared in rabbits and sheep to rat liver ribosomes, ribosomal subunits, and to mixtures of proteins from the particles. The antisera were characterized by quantitative immunoprecipitation, by passive hemagglutination, by immunodiffusion on Ouchterlony plates, and by immunoelectrophoresis. While all the antisera contained antibodies specific for ribosomal proteins, none had precipitating antibodies against ribosomal RNA. Rat liver ribosomal proteins were more immunogenic in sheep than rabbits, and the large ribosomal subunit and its proteins were more immunogenic than those of the 40S subparticle. Antisera specific for one or the other ribosomal subunit could be prepared; thus it is unlikely that there are antigenic determinants common to the proteins of the two subunits. When ribosomes, ribosomal subunits, or mixtures of proteins were used as antigens the sera contained antibodies directed against a large number of the ribosomal proteins.Abbreviations TP total proteins—used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixtures of all the proteins from 80S ribosomes - TP60 the proteins from 60S subunits - TP40 the proteins from 40S particles     

Structural changes in ribosomes during development

Basic ribosomal proteins, extracted from immature erythrocytes of both pre-metamorphic and adult bullfrogs (Rana catesbeiana), were characterized on urea-polyacrylamide gels. Ribosomes from larval and adult organisms display quantitative and qualitative differences in the banding patterns of their basic ribosomal proteins. Such alterations in ribosomal structure may be important in the control of gene expression during development.     

Tracing the evolution of RNA structure in ribosomes

The elucidation of ribosomal structure has shown that the function of ribosomes is fundamentally confined to dynamic interactions established between the RNA components of the ribosomal ensemble. These findings now enable a detailed analysis of the evolution of ribosomal RNA (rRNA) structure. The origin and diversification of rRNA was studied here using phylogenetic tools directly at the structural level. A rooted universal tree was reconstructed from the combined secondary structures of large (LSU) and small (SSU) subunit rRNA using cladistic methods and considerations in statistical mechanics. The evolution of the complete repertoire of structural ribosomal characters was formally traced lineage-by-lineage in the tree, showing a tendency towards molecular simplification and a homogeneous reduction of ribosomal structural change with time. Character tracing revealed patterns of evolution in inter-subunit bridge contacts and tRNA-binding sites that were consistent with the proposed coupling of tRNA translocation and subunit movement. These patterns support the concerted evolution of tRNA-binding sites in the two subunits and the ancestral nature and common origin of certain structural ribosomal features, such as the peptidyl (P) site, the functional relay of the penultimate stem helix of SSU rRNA, and other structures participating in ribosomal dynamics. Overall results provide a rare insight into the evolution of ribosomal structure.     

The ribosomes of Drosophila

Summary The assembly of proteins and RNA into mature ribosomal subunits has been studied in Drosophila cell cultures by pulse-chase experiments. Pulse labeled rRNA has a transit time of 3 h, while the transfer of ribosomal protein occurs completely within 30 min. Inhibition of protein synthesis by cycloheximide results in an almost immediate cessation of ribosome assembly, a result which indicates that no large pool of free ribosomal proteins exists in the cell. Substituting pre-ribosomal RNA with the analogue 5-fluorouridine (5-FU) results in a cessation of ribosome maturation. Under these conditions at least three large subunit proteins continue to accumulate on pre-existing cytoplasmic subunits, indicating an exchange. A portion of ribosomal subunit proteins synthesized in the presence of 5-FU can be recovered in cytoplasmic subunits once the effect of 5-FU has been reversed. This is most easily interpreted in terms of their stabilization on substituted pre-rRNA within the nucleolus, and subsequent utilization on unsubstituted RNA.Work supported by a grant from the NIH (GM 22866)     

Approaching the molecular structure of ribosomes

Fifteen forms of three-dimensional crystals and three forms of two-dimensional sheets from ribosomal particles have been grown. In all cases only biologically active particles could be crystallized, the crystalline material retaining its integrity and biological activity for months. Cryastallographic data have been collected from crystals of 50 S ribosomal subunits, using synchrotron radiation, at temperatures between 19 and -180 degree C. Although at around 0 degrees C in the synchrotron X-ray beam the crystals rapidly lose their high-resolution reflections, at cryo-temperatures hardly any radiation damage occurs over long periods, and a complete set of diffraction data to about 6 A resolution could be collected from a single crystal. Heavy-atom clusters were used for soaking as well as for specific binding to the surface of the ribosomal subunits prior to crystallization. The 50 S ribosomal subunits from a mutant of Bacillus stearothermophilus which lacks the ribosomal protein BL11 crystallize isomorphously with the native form. Models of the entire 70 S ribosome and of the 50 S subunit have been reconstructed from two-dimensional sheets at 47 and 30 A, respectively. These models demonstrate the overall shape of the particles, the contact areas between large and small subunits, the space where protein biosynthesis may take place and a tunnel through the 50 S subunit which could provide a path for the nascent polypeptide chain.     

Properties of human mitochondrial ribosomes

Mammalian mitochondrial ribosomes (55S) differ unexpectedly from bacterial (70S) and cytoplasmic ribosomes (80S), as well as other kinds of mitochondrial ribosomes. Typical of mammalian mitochondrial ribosomes, the bovine mitochondrial ribosome has been developed as a model system for the study of human mitochondrial ribosomes, to address several questions related to the structure, function, biosynthesis and evolution of these interesting ribosomes. Bovine mitochondrial ribosomal proteins (MRPs) from each subunit have been identified and characterized with respect to individuality and electrophoretic properties, amino acid sequence, topographic disposition, RNA binding properties, evolutionary relationships and reaction with affinity probes of ribosomal functional domains. Several distinctive properties of these ribosomes are being elucidated, including their antibiotic susceptibility and composition. Human mitochondrial ribosomes lack several of the major RNA stem structures of bacterial ribosomes but they contain a correspondingly higher protein content (as many as 80 proteins), suggesting a model where proteins have replaced RNA structural elements during the evolution of these ribosomes. Despite their lower RNA content they are physically larger than bacterial ribosomes, because of the 'extra' proteins they contain. The extra proteins in mitochondrial ribosomes are 'new' in the sense that they are not homologous to proteins in bacterial or cytoplasmic ribosomes. Some of the new proteins appear to be bifunctional. All of the mammalian MRPs are encoded in nuclear genes (a separate set from those encoding cytoplasmic ribosomal proteins) which are evolving more rapidly than those encoding cytoplasmic ribosomal proteins. The MRPs are imported into mitochondria where they assemble coordinately with mitochondrially transcribed rRNAs into ribosomes that are responsible for translating the 13 mRNAs for essential proteins of the oxidative phosphorylation system.     

Resolution and identification of ribosomes from mycobacteria

Ribosomes were isolated from mycobacteria (BCG) by differential centrifugation by use of a modification of Nirenberg's procedure. Individual components of the ribosomal spectrum were resolved in linear sucrose density gradients. Sedimentation constants of the individual components were determined by analytical ultracentrifugation. Ribosomes from escherichia coli labeled with (14)C-uracil were used as markers for an independent confirmation of the identity of individual peaks. The typical ribosomal spectrum of 70S units and 50S and 30S subunits was obtained in tris(hydroxymethyl)aminomethane buffer with 0.01 m Mg(CH(3)COO)(2). This corresponds to the ribosomal spectrum obtained in E. coli under comparable conditions. In 0.0001 m Mg(2+), the 70S units were dissociated to 50S and 30S particles. Pancreatic ribonuclease produced no significant changes in main ribosomal components. The isolation techniques used in this work precluded the recognition of polyribosomes.     

Absence of ribosomes in Capsicum chromoplasts

Ribosome development was followed by electron microscopy and gel electrophoresis of ribosomal (r)RNAs in the plastids of fully expanded fruits of Capsicum annuum L. during ripening. Chloroplasts from young Capsicum leaves were used as a structural and electrophoretic standard. Four stages were distinguished on the basis of colour changes during fruit ripening. Chloroplasts of the green fruit had a lower content of 16S and 23S rRNAs than leaf chloroplasts. They contained only a few ribosomes, some more discrete ribosomal particles, and the contrast of ribosomal structures was faint. From the outset of ripening, most of the ribosomal structures in the plastid stroma disappeared. A continuous decrease in plastid rRNAs occurred during ripening. Fully differentiated chromoplasts of the red fruit did not contain rRNAs or ribosomes. Throughout plastid development, DNA nucleoids were evident and there was only a small decrease in the DNA peak on electrophoretograms. The loss of ribosomes during the chloroplast-to-chromoplast conversion in Capsicum fruit is discussed in relation to the variations in pigments and enzymic systems in both plastid types.Abbreviations Developmental stages of leaves and fruits: A four-week-old green leaf - B green fruit - C brownish fruit - D orange fruit - E red fruit - ptRNA, DNA plastid RNA - DNA; rRNA ribosomal RNA     

Proteins of mammalian mitochondrial ribosomes.

The bovine mitochondrial system is being developed as a model system for studies on mammalian mitochondrial ribosomes. Information is emerging on the structural organization and RNA binding properties of proteins in these mitochondrial ribosomes. Unexpectedly, these ribosomes appear to interact directly with GTP, via a high affinity binding site on the small subunit. Despite major differences in their RNA content and physical properties, mammalian mitochondrial and cytoplasmic ribosomes contain about the same number of proteins. The proteins in each kind of ribosome have a similar size distribution, and both sets are entirely coded by nuclear genes, raising the possibility that these different ribosomes may contain the same set of proteins. Comparison of bovine mitochondrial and cytoplasmic r-proteins by co-electrophoresis in two-dimensional gels reveals that most of the cytoplasmic ribosomal proteins are more basic than the mitochondrial ribosomal proteins, and that none are co-migratory with mitochondrial ribosomal proteins, suggesting that the proteins in the two ribosomes are different. To exclude the possibility that the electrophoretic differences result only from post-translational modification of otherwise identical proteins, antibodies against several proteins from the large subunit of bovine mitochondrial ribosomes were tested against cytoplasmic ribosomes by solid phase radioimmunoassay and against cytoplasmic ribosomal proteins on Western blots. The lack of cross-reaction of these antibodies with cytoplasmic r-proteins suggests that mitochondrial ribosomal proteins have different primary structures and thus are most likely encoded by a separate set of nuclear genes.     

Quantitative analysis of the protein composition of yeast ribosomes

The molecular weights of the individual yeast ribosomal proteins were determined. The ribosomal proteins from the 40-S subunit have molecular weights ranging from 11 800 to 31 000 (average molecular weight = 21 300). The molecular weights of the 60-S subunit proteins range from 10 000 to 48 400 (average molecular weight = 21 800). Stoichiometric measurements, performed by densitometric scanning on ribosomal proteins extracted from high-salt dissociated subunits revealed that isolated ribosomal subunits contain, besides some protein species occurring in submolar amounts, a number of protein species which are present in multiple copies: S13, S27, L22, L31, L33, L34 and L39. The mass fractions of the ribosomal proteins which were found to be present on isolated ribosomes in non-unimolar amounts, were re-examined by using an isotope dilution technique. Applying this method to proteins extracted from mildely isolated 80-S ribosomes, we found that some protein species such as S32, S34 and L43 still are present in submolar amounts. On the other hand, however, we conclude that some other ribosomal proteins, in particular the strongly acidic proteins L44 and L45 get partially lost during ribosome dissociation. Proteins L44/L45 appears to be present on 80-S ribosomes in three copies.     

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.     

Reactivity of proteins in ribosomes from Saccharomyces cerevisiae with trypsin

The susceptibility of 40S and 60S ribosomal subunits from Saccharomyces cerevisiae to digestion with varying concentrations of trypsin was studied by two-dimensional electrophoresis and quantitative measurements of the protein remaining with the ribosomal particles after trypsin treatment. Proteins from both subunits can be classified into three groups according to their rate of digestion by trypsin. These results are in good agreement with those obtained on the order of ribosomal assembly in vivo, i.e., proteins which are most susceptible to trypsin digestion have been shown to associate with the ribosomal particles at a relatively late stage of ribosome assembly.