Isolation of stable ribosomal subunits from spores of Bacillus cereus.

Analyses of ribosomes extracted from spores of Bacillus cereus T by a dryspore disruption technique indicated that previously reported defects in ribosomes from spores may arise during the ribosome extraction process. The population of ribosomes from spores is shown to cotain a variable quantity of free 50S subunits which are unstable, giving rise to slowly sedimenting particles in low-Mg2+ sucrose gradients and showing extremely low activity in in vitro protein synthesis. The majority of the ribosomal subunits in spores, obtained by dissociation of 70S ribosomes and polysomes, are shown to be as stable as subunits from vegetative cells, though the activity of spore polysomes was lower than that of vegetative ribosomes. In spite of the instability and inactivity of a fraction of the spore's ribosomal subunits, the activity of the total population obtained from spores by the dry disruption technique was 32% of vegetative ribosome activity, fivefold higher than previously obtained with this species. The improvement in activity and the observed variability of subunit destabilization are taken as evidence for partial degradation of spore ribosomes during extraction.     

Membrane-bound ribosomes in kidney: methods of estimation and effect of compensatory renal growth

Membrane-bound ribosomes are thought to secrete protein for export and free ribosomes to secrete protein for intracellular use. The proportion of the total ribosomes that is bound to membranes in normal mouse kidneys has been estimated by three different methods, and the results have been compared with those obtained by a fourth method used by us previously. The most valid estimates appear to be those obtained (a) by comparison of radioactivity in peaks representing free and membrane-bound ribosomes on linear sucrose gradients after labeling for 24 hr with ¹⁴C-orotic acid, and (b) by measurements of optical density in free and bound ribosomes that had been separated by centrifugation on discontinuous gradients of 0.5 M/2.0 M sucrose. Analyses by these methods show that about 20–25% of the ribosomes in a postnuclear supernatant prepared from mouse kidneys, but only 10–15% of the ribosomes in a post-mitochondrial supernatant, are membrane-bound. About 75% of the bound ribosomes sediment as polysomes of many different sizes. The proportion of membrane-bound ribosomes and their aggregation into polysomes were unchanged in kidneys undergoing compensatory hypertrophy after removal of the opposite kidney. These experiments show that, unlike liver, kidney has a predominance of free ribosomes compared to bound ribosomes; those ribosomes that are membrane-bound do not become free during compensatory renal growth.     

A novel chromatography system to isolate active ribosomes from pathogenic bacteria

We have developed a novel chromatography for the rapid isolation of active ribosomes from bacteria without the use of harsh conditions or lengthy procedures that damage ribosomes. Ribosomes interact with an alkyl linker attached to the resin, apparently through their RNA component. Examples are given with ribosomes from Escherichia coli, Deinococcus radiodurans, and with clinical isolates of Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). The ribosomes obtained by this method are unusually intact, so that highly active ribosomes can now be isolated from the clinical isolates, enabling significantly improved in vitro functional assays that will greatly assist the discovery and development of new ribosomally targeted antibiotics.     

Isolation and purification of bacterial ribosomes from endotoxin using polyethylene glycol

The difficulties arising in the study of the immunogenicity of bacterial ribosomes and in their possible use as vaccines are due to the fact that preparative ultracentrifugation, constituting a necessary stage in most of the methods used for the isolation of ribosomes, has a low productive capacity. To develop a more effective method for obtaining Shigella ribosomal vaccines, an attempt to use the method of precipitation with 10% polyethylene glycol (PEG), proposed by Expert-Bezan?on et al., has been made. The serological determination of O antigen has shown that nearly contained in the supernatant fluid S-30 can be detected in precipitated ribosomes. Taking into account the wide spectrum of the biological activity of bacterial endotoxin, it must be removed from the vaccine. The study has revealed that precipitation by means of ethanol (15-35%), low pH (4,2-4,7) and PEG (4-8%) can be used for this purpose. In accordance with the chosen method, the clarified material obtained by precipitation with 10% PEG is fractionated by means of 5% PEG which causes the complete precipitation of ribosomes, thus leaving endotoxin in the solution. Centrifugation in the density gradient of saccharose and electron microscopy have demonstrated that ribosomes isolated by this method possess typical sedimentation properties and structure. The yield of ribosomes is 3 times greater than that obtained by ultracentrifugation. Fractionation with PEG may be used as the method of the mass production of ribosomal vaccines.     

Isolation of 80S ribosomes from spores of Aspergillus fumigatus and their antigenicity in rabbits

Ribosomes were obtained from spores of Aspergillus fumigatus by mechanical disruption and differential centrifugation. The initial preparation (crude ribosomes) contained spore components which appeared to be broken fragments of plasmalemma with or without organelles. Purified ribosomes free of membranous material were prepared by gel filtration chromatography on Sepharose CL-4B. Monomeric 80S ribosomes consisting of 40% protein and 60% RNA, and morphologically characteristic of fungal ribosomes were isolated.Serological reactions in sera from rabbits injected with crude or purified ribosomes were similar indicating that the purification process did not change or eliminate antigens that stimulated antibody detectable either by indirect hemagglutination (IHA) or in gel precipitin tests.     

Purification of eukaryotic ribosomes by isopycnic centrifugation in sucrose

A simple and inexpensive procedure for the isolation and purification of ribosomes from eukaryotes is described. The method avoids pelleting of ribosomes at high centrifugal forces and involves isopyenic centrifugation of the post-mitochrondrial supernatant in sucrose, precipitation of ribosomes with 10% polyethylene glycol, and zonal sucrose gradient centrifugation. The ribosomes obtained in this way are very pure and thus especially suited for the measurements of physical properties. The isopycnic centrifugation can also be used for the purification of other macromolecules and is only limited by a maximum density of sucrose of 1.40 g/cm³ obtained at the bottom of the centrifugation tubes.     

G-418, an elongation inhibitor of 80 S ribosomes

The mode of action of the aminoglycoside G-418 was studied in wheat-germ, cell-free translation systems programmed with rat-liver polyadenylated RNA. Incorporation of amino acids into protein was effectively inhibited by G-418 in the microM concentration range. The inhibition pattern obtained was not uniform. The synthesis of polypeptides with higher molecular weights was more inhibited than that of smaller polypeptides. An identical inhibition pattern within a similar range of concentrations was obtained with cycloheximide, a known elongation inhibitor. Translation activity was abolished when the wheat-germ 80 S ribosomes were removed and could be partially reconstructed upon addition of the ribosomes. Incubation with G-418 prior to isolation yielded ribosomes defective in their reconstruction ability. The inhibition pattern was not uniform and exhibited again the same relationship between the size of a polypeptide and the extent of inhibition of its synthesis. Therefore, we suggest that in wheat-germ, cell-free translation systems G-418 affects the 80 S ribosomes and inhibits the elongation cycle.     

Regulation of polyribosome formation and protein synthesis in the uterus. Isolation of cytoplasmic ribonucleoprotein particles and the principal properties of the cell-free protein-synthesizing system

1. Three procedures for isolating ribonucleoprotein particles from the cytoplasmic fraction of rat-uterus homogenates are described. By procedure 1, ribonucleoprotein particles were isolated in the presence of 5mm-Mg(2+) and 25mm-K(+), and the postmitochondrial supernatant fraction was made to 1.3% (w/v) in potassium deoxycholate. About 50% of the RNA and protein of the microsomal fraction was recovered in the monomeric ribosomes isolated. By procedure 2, ribonucleoprotein particles were isolated in the presence of 10mm-Mg(2+) and 0.1m-K(+), and in the absence of detergent. The ribosomes obtained were primarily polymeric, but recovery of microsomal RNA and protein was only 32%. By procedure 3, ribonucleoprotein particles were isolated according to procedure 1 but without the use of detergent. A mixture of polymeric and monomeric ribosomes was obtained, and the recovery of microsomal RNA and protein was about 60%. 2. Uterine polymeric and monomeric ribosomes, isolated by procedure 3 and designated ;polyribosomal preparation', were examined for protein-synthesizing capabilities. The principal properties of the cell-free protein-synthesizing system containing the polyribosomal preparation are described. The efficiency of amino acid incorporation in the complete system incubated for 30min. and containing the polyribosomal preparation was found to be either 2.5 molecules of [(14)C]leucine or 2.2 molecules of [(14)C]-valine incorporated/ribosome. Assay of the preparation in the complete cell-free system containing 10mm-sodium fluoride indicated that 40% of the incorporation activity is a result of initiation of new polypeptide chains and 60% is due to completion of previously existing chains. Monomeric ribosomes obtained by various treatments of the polyribosomal preparation with sodium fluoride, ribonuclease and potassium deoxycholate had decreased incorporation activity in the cell-free system. However, monomeric ribosomes obtained by treatment with sodium fluoride only had an incorporation activity 50% greater than that of monomers obtained by treatment with ribonuclease only. 3. The results indicate that uterine polymeric and monomeric ribosomes are sites of amino acid incorporation in vivo and in vitro. It is concluded that most polymeric and monomeric ribosomes occurring in the cytoplasmic fraction of the uterus are free and unattached to membranes, and that the polyribosomes are relatively unstable.     

The role of ribosomal ribonucleic acid in the structure and function of mammalian brain ribosomes

In order to resolve the functional role of intact rRNA in polypeptide chain elongation mouse brain ribosomes were treated with dilute pancreatic or T(1) RNAase (ribonuclease). After RNAase treatment, several physical-chemical properties as well as the functional activity of the ribosomes were measured. RNAase treatment resulted in the extensive hydrolysis of both 18S and 28S rRNA; however, the sedimentation properties of mono-ribosomes were unaltered and more than 90% of the relatively low-molecular-weight RNA fragments remained associated with ribosome particles. Analysis of the ability of RNAase-treated ribosomes to participate in cell-free protein synthesis showed that ribosomes with less than 2% intact rRNA retained more than 85% of their activity in polyphenylalanine incorporation. Proof that the incorporation of phenylalanine by ribosomes with hydrolysed rRNA actually represented active translocation was obtained by the effective inhibition of incorporation by diphtheria toxin. In addition, the oligopeptide products of protein synthesis could be identified by BD (benzoylated diethylaminoethyl)-cellulose column chromatography. Analysis of the size distribution of oligopeptides synthesized by normal and RNAase-treated ribosomes showed no significant differences which indicated that there was no change in the proportion of ribosomes engaged in protein synthesis. Thus strong RNA-protein and protein-protein interactions must serve to maintain the functional integrity of ribosomes even when the rRNA is extensively degraded. The ability of the enzyme-treated ribosomes to efficiently incorporate amino acids clearly demonstrated that ;intact' rRNA is not required for protein-synthetic activity.     

Rapidly labelled ribonucleic acid from rat liver. Studies in the attachment to ribosomes and stimulation of the incorporation of amino acids into polypeptides in cell-free systems

1. Rapidly labelled RNA from rat liver, either as a complex with DNA (m-RNA-DNA) or with ribosomal RNA (m-RNA-RNA) binds to ribosomes in the polysome region. No binding could be demonstrated with ribosomal RNA or native DNA from Bacillus subtilis. 2. With ribosomes from rat liver, Escherichia coli or hepatoma the m-RNA-DNA stimulated incorporation of amino acids with rat-liver ribosomes only, whereas the m-RNA-RNA complex was effective with ribosomes from E. coli or the hepatoma. 3. Polyuridylic acid was effective as messenger RNA with all three ribosomes but much greater stimulation was obtained with ribosomes from E. coli and the hepatoma. 4. The degree of incorporation of phenylalanine with polyuridylic acid and ribosomes from a hepatoma was decreased by about 50% when ribosomal RNA was present.     

IN VlTRO BINDING OF PHENYLALANYL-tRNA TO NEONATAL AND ADULT MOUSE BRAIN RIBOSOMES

The ability of brain ribosomes, isolated from mice of various ages, to bind phenylalanyl-tRNA was measured under various reaction conditions. In the presence of template RNA (polyuridylic acid) the binding could be measured by both enzymic and non-enzymic assays. In general, the binding requirements for the brain system were similar to those previously described for microbial and eukaryotic systems. Although previous studies have shown that ribosomes obtained from increasingly older mow brain tissue were less active in polyphenylalanine synthesis, no significant differences in phenylalanyl-tRNA binding to polysome complexes could be detected. The binding of phenylalanyl-tRNA by ribosomes isolated from both neonatal and mature mouse brain tissue was similar with regard to GTP and polyuridylic acid dependence, magnesium ion concentration and reaction kinetics. Similar binding of phenylalanyl-tRNA by young and mature brain ribosomes was also measured with ribonucleoprotein particles previously stripped with puromycin. The results are discussed in light of the rapid alteration of macromolecular synthesis during postnatal brain development and the possible role of the interaction between ribosomes and tRNA.     

Identification of helical polyribosomes in sections of mature skeletal muscle fibers

Summary The localization and configurations of ribosomes in mature white skeletal muscle fibers of the rat were investigated. Differential visualization of ribosomes and glycogen granules was obtained by fixation with glutaraldehyde only and staining of the sections with uranyl acetate. Ribosomes are then electron dense and glycogen granules electron transparent. Their identity was ascertained by selective extractions of ribonucleic acid and polysaccharide.The vast majority of the ribosomes is not membrane-bound. They are located intermyofibrillarly (predominantly at the level of the I-bands), beneath the sarcolemma, and in the paranuclear cones of sarcoplasm. Occasionally short stretches of granular reticulum occur, often as characteristic double walled vesicles with ribosomes on the inner membrane only.Three main types of polysomal configurations are observed: rosettes of 4 to 6 ribosomes, helical arrays, and whorls of up to about 25 probably membrane-bound ribosomes. The average number of ribosomes in the extended helical configurations is estimated to be about 60. It is argued that these helices represent the polysomes instrumental in the synthesis of the large subunits of myosin. It is emphasized that helical polyribosomes are by no means typical of striated muscle, but probably represent a common configuration of large free polysomes.With the technical assistance of Tineke J. Hoogenboezem.     

Polysome stability in relaxed and stringent strain of Escherichia coli during amino acid starvation

The influence of amino acid starvation on polysome content was examined in relaxed and stringent strains of Escherichia coli which were isogenic for the RC locus. No difference was observed between the polysome profiles obtained from two different sets of stringent and relaxed strains starved for the same amino acid. In both relaxed and stringent strains, starvation for amino acids other than methionine resulted in only a slight breakdown of polysomes with a concomitant increase of 70S ribosomes. However, starvation for methionine in both RC stringent and relaxed strains of E. coli resulted in a more extensive degradation of polysomes and accumulation of 70S ribosomes. The 70S ribosomes obtained as a result of methionine starvation were more sensitive to degradation to 50 and 30S subunits in 10(-3)m Mg(2+) than 70S monomers obtained either by degradation of polysomes with ribonuclease or by starvation of cells for amino acids other than methionine. The 70S ribosomes from methionine starvation were similar (sensitivity to 10(-3)m Mg(2+)) to 70S ribosomes obtained from cells in which initiation of protein synthesis had been prevented by trimethoprim, an inhibitor of formylation. Since N-formyl-methionyl-transfer ribonucleic acid is required for initiation, the 70S ribosomes obtained in both methionine-starved and trimethoprim-treated cells must result from association of 50 and 30S subunits for reasons other than reinitiation. These results suggest that the level of ribonucleic acid synthesis does not influence the distribution of ribosomes in the polysome profile and vice versa.     

Dissociation of mitochondrial ribosomes of neurospora crassa by a bacterial dissociation factor

From ribosomes of Escherichia coli a protein factor can be obtained that promotes dissociation of bacterial ribosomes into subunits. Incubation of mitochondrial ribosomes from Neurospora crassa with the bacterial dissociation factor also leads to the formation of subunits. Under the same conditions no dissociation of cytoplasmic ribosomes from Neurospora crassa was observed.     

Stoichiometry of GTP hydrolysis during peptide synthesis on the ribosome. I. Factor-independent GTPase and ATPase of ribosomal preparations

It has been found that preparations of Escherichia coli (MRE-600) ribosomes can display GTPase and ATPase activities independent of elongation factors EF-Tu and EF-G. The GTPase and ATPase are localized on ribosomal 50S subparticles, whereas 30S subparticles are free of the activities and do not stimulate them upon association with the 50S subparticles to form complete ribosomes. The GTPase and ATPase can be removed from the ribosomes and their 50S subparticles by treatment with 1 M NH4Cl or 50% ethanol in the cold. Ribosomal preparations freed from the factor-independent GTPase and ATPase retain their basic functional features. The data obtained do not permit to solve finally whether the factor-independent GTPase and ATPase revealed are components of ribosomes or represent a contamination rather firmly bound to the ribosomes. However, in any case this finding can contribute to an uncoupled hydrolysis of GTP and should be considered when studying the stoichiometry of triphosphate expenditure in the process of ribosomal protein synthesis.     

Experimental changes in the amount of maternally stored ribosomes affect the translation efficiency of ribosomal protein mRNA in Xenopus embryo

The amount of maternal free ribosomes in developing Xenopus embryos has been experimentally modified; an increase was obtained by microinjection of purified ribosomes into fertilized eggs, and a decrease was induced by treatment with a drug which reduces the amount of free ribosomes. The effect of this manipulation on the partition of the ribosomal protein mRNA (rp-mRNA) was analyzed during embryo development; it was observed that when ribosomes available for translation are in excess, polysome loading with rp-mRNA decreases. Conversely, when ribosomes are scarce, polysome loading of rp-mRNA increases. These experiments, which artificially stress events observed in the course of development, indicate that there is a relationship between the availability of ribosomes in the cells and the utilization of rp-mRNA for synthesis of ribosomal proteins, as already suggested by previous observations on r-protein synthesis during embryogenesis.     

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.     

The contribution of the zinc-finger motif to the function of Thermus thermophilus ribosomal protein S14

In the crystal structure of the 30S ribosomal subunit from Thermus thermophilus, cysteine 24 of ribosomal protein S14 (TthS14) occupies the first position in a CXXC-X12-CXXC motif that coordinates a zinc ion. The structural and functional importance of cysteine 24, which is widely conserved from bacteria to humans, was studied by its replacement with serine and by incorporating the resulting mutant into Escherichia coli ribosomes. The capability of such modified ribosomes in binding tRNA at the P and A-sites was equal to that obtained with ribosomes incorporating wild-type TthS14. In fact, both chimeric ribosomal species exhibited 20% lower tRNA affinity compared with native E. coli ribosomes. In addition, replacement of the native E. coli S14 by wild-type, and particularly by mutant TthS14, resulted in reduced capability of the 30S subunit for association with 50S subunits. Nevertheless, ribosomes from transformed cells sedimented normally and had a full complement of proteins. Unexpectedly, the peptidyl transferase activity in the chimeric ribosomes bearing mutant TthS14 was much lower than that measured in ribosomes incorporating wild-type TthS14. The catalytic center of the ribosome is located within the 50S subunit and, therefore, it is unlikely to be directly affected by changes in the structure of S14. More probably, the perturbing effects of S14 mutation on the catalytic center seem to be propagated by adjacent intersubunit bridges or the P-site tRNA molecule, resulting in weak donor-substrate reactivity. This hypothesis was verified by molecular dynamics simulation analysis.     

Rapid peptide bond formation on isolated 50S ribosomal subunits

The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.     

Ribosome inactivation by the toxic lectins abrin and ricin. Kinetics of the enzymic activity of the toxin A-chains.

A sensitive test system for toxin-treated ribosomes was worked out by treating rabbit reticulocyte ribosomes with abrin A-chain, ricin A-chain or ricinus agglutinin A-chain, adding neutralizing amounts of specific antitoxins and testing for polyphenylalanine-synthesizing activity in a system where the concentration of elongation factors and ribosomes were varied. The strongest inhibition was obtained in the presence of low concentrations of elongation factor (EF-2). The activity of the ribosomes decreased with time of incubation with the toxin A-chains. Addition of anti-toxins stopped further inactivation. In systems containing untreated and toxin-treated ribosomes the ability to polymerize phenylalanine was proportional to the concentration of untreated ribosomes. There was a linear relationship between toxin A-chain concentration and the number of ribosomes inactivated per minute. The inactivation rate increased with temperature, and the estimated activation energy was 10.6 kcal (44.3 kJ). Linewaver-Burk plots of the data obtained by incubating various ribosome concentrations with toxins indicated a molecular activity of about 1500 ribosomes/minute for abrin and ricin A-chains and 100 ribosomes/minute for ricinus agglutinin A-chain. The apparent Michaelis constant was 0.1-0.2 muM for all three A-chains. The activity of the A-chains in the intact cell is discussed.