Free 70S Ribosomes Research Articles

Formation of 100S ribosomes in Staphylococcus aureus by the hibernation promoting factor homolog SaHPF

In the stationary growth phase of Escherichia coli, the 70S ribosomes are dimerized by the ribosome modulation factor (RMF) and hibernation promoting factor (HPF) proteins to form 100S ribosomes, which lose translational activity. In this study we found 100S ribosomes in the gram-positive bacterium Staphylococcus aureus, which has an HPF homolog (named SaHPF) but no RMF homolog. Unlike in E. coli, 100S ribosomes exist in all growth phases of S. aureus, with the highest levels at the transition from the exponential phase to the stationary phase. To find the key factors involved in 100S formation, we analyzed proteins associated with crude ribosomes using radical-free and highly reducing 2-D PAGE and MALDI TOF/MS. Only the SaHPF levels changed in parallel with the changes in 100S levels. SaHPF bound preferentially to 70S components in 100S ribosomes, with a molar ratio of 1 : 1 relative to the 70S, but some SaHPF was also detected in free 70S ribosomes. High-salt washing of the crude ribosomes released SaHPF and dissociated the 100S ribosomes to their 70S components. When these 70S components were incubated with purified SaHPF in vitro, they re-associated to form 100S. These results suggest that SaHPF is a key protein involved in 100S ribosome formation in S. aureus.

The effects of nuclear mutation on chloroplast development

Under greenhouse conditions the dark green wild type (su/su) tobacco grows 2-3 times faster than the yellow mutant (Su/su) and contains five-fold more chlorophyll. On a fresh weight basis, however, both genotypes contain similar amounts of RuBPCase and fraction 11 protein in approximately equal proportion and have similar levels of 70s and 80s ribosomes. When seedlings are cultured on agar medium supplemented with sucrose and equal concentrations of IAA and kinetin or kinetin alone, a drastic reduction of RuBPCase and free 70s ribosomes, but not of chlorophyll content, were observed. Moreover, albino (Su/Su) seedlings developed on supplemented media still contain appreciable amounts of RuBPCase and free 70s ribosomes although chlorophyll levels are extremely low indicating no correlation between RuBPCase and chlorophyll content. RuBPCase crystallized from both wild type and yellow mutant plants seem to have identical composition and structure when examined by isoelectric focusing, amino acid analysis or peptide mapping techniques. The slow-growing yellow mutant is apparently deficient only in chlorophyll of the light harvesting chlorophyll-protein complex but with no alteration of the protein moiety or chlorophyll a/b ratio.

Hormonal Effects on the Biosynthesis of Tobacco RuBPCase in vitro

Abstract1. The three genotypes, Su/Su, Su/su, su/su, of John Williams Broadleaf tobacco (N. tabacum) grew equally well on Murashige-Skoog medium. On a fresh weight basis, the heterozygous (Su/su) yellow mutant and the homozygous (su/su) wild type plants contain similar amounts of RuBPCase and Fraction II protein in roughly equal proportions, but with a somewhat reduced amount of 70S and 80S free ribosomes. The homozygous albino (Su/Su) plants contain appreciable amounts of RuBPCase and free 70S ribosomes, although the chlorophyll levels are extremely low. In the case of the yellow and albino forms, there appears to be no correlation between RuBPCase and chlorophyll content.`2. When equal concentrations (1 mg/l) of kinetin and indole-3-acetic acid (IAA), or kinetin alone, were added to the medium, a drastic reduction of RuBPCase and disappearance of free 70S ribosomes were observed in the yellow mutant and wild type plants, whereas the effect on the albino plants was not significant.

The chemical modification of Escherichia coli ribosomes with methyl p-nitrobenzenesulfonate. Evidence for the involvement of a histidine residue in the functioning of the ribosomal peptidyl transferase.

Free 70S ribosomes and 70S ribosome – AUG – f Met-tRNA complexes were reacted, separately with methyl p-nitrobenzenesulfonate. This reagent inhibits the peptidyl transferase activity of 70S ribosome – AUG – fMet-tRNA but not of free 70S ribosomes. The data suggest that methyl p-nitrobenzenesulfonate reacts with a unique site which is a part of the ribosomal peptidyl transferase.

Protein deficient chloroplast ribosomes in a yellow mutant of Chlamydomonas reinhardii

Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analyzed and compared by two-dimensional gel electrophoresis. The mixothrophically grown yellow-76 mutant differs from wild-type cells in lowered chlorophyll content and photosynthetic activity per chlorophyll unit. The latter is connected with the decreased activity of the ribulose-I,5-diphosphate-carboxylase enzyme. Analytical ultracentrifugation of cell extracts shows a normal amount of free 70S ribosomes and 50S subunit in the mutant cells. Two-dimensional gel electrophoresis shows considerable alterations in the protein composition of the 70S ribosomes of the mutant. Two proteins are absent from the electrophoretograms of the yellow-76 mutant, and seven proteins are present in reduced amounts. The genetical analysis shows a Mendelian pattern of inheritance, indicating that protein alterations presumably are localized in nuclear DNA.

Altered chloroplast ribosomal proteins in a yellow mutant of Chlamydomonas reinhardii

Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analysed and compared by two-dimensional gel electrophoresis. Mixothrophycally grown yellow-27 mutant differs from wild-type cells in lowered chlorophyll content and grana formation of the chloroplast. Analytical ultracentrifuge analyses of cell extracts show a reduced amount of free 70S ribosomes and increased level of 50S subunits in the mutant cells. Similar results were obtained by electronmicroscopical method. Two-dimensional gel electrophoresis shows alterations in protein composition of 70S ribosomes of the mutant. Two proteins of 70S ribosomes have been altered. One of them with high molecular weight is practically absent while there is an additional, intensively stained spot in the mutant. Since the mutation is inherited in a non-Mendelian manner it is possible that the protein alterations in 70S ribosome are localized in the chloroplast DNA.

The Formation of Guanosine-Nucleotide . Elongation-Factor-G . Ribosome Complexes on Free 70-S Ribosomes, 50-S Subunits, and Polysomes. A Comparative Study

The ability of free 70-S ribosomes, 50-S subunits and purified, endogenous Escherichia coli polysomes to form complexes with elongation factor G (EF-G) and different concentrations of guanosine nucleotides has been examined. GTP (plus fusidic acid) has been used with all these types of ribosomal particles and GDP (plus the antibiotic), guanyl-5′-yl methylenediphosphonate (Guo-5′-P2-CH2-P) and guanyl-5′-yl imidodiphosphate (Guo-5′-P2-NH-P) with free 70-S ribosomes. In every case the data, obtained with the nitrocellulose membrane filtration technique and under saturating concentrations of EF-G and fusidic acid (when present), fell on one straight line in Scatchard plots, thus indicating that the sites involved in guanosine nucleotide binding are homogeneous and lack cooperative interactions. With 70-S ribosomes between 0.6 and 0.75 molecule of nucleotide were bound per ribosome at saturating concentrations of guanosine nucleotide (3–10 μM). Assuming that about one-third of ribosomes in the preparations were inactive, this result suggests that with each of the four guanosine nucleotides one molecule of this ligand was bound per ribosome. The apparent affinity constants Ka were: for GTP = 7.7 × 106 M−1, for GDP = 1.3 × 107 M−1, for Guo-5′-P2-CH2-P= 5.0 × 106 M−1 and for Guo-5′-P2-NH-P= 1.9 × 106 M−1. 50-S subunits, under the conditions used (10 mM Mg2+ and 65 mM NH+4) and at saturating concentrations of GTP, only complexed about 0.35 molecule of nucleotide per subunit and the apparent KGTPa was 1.2 × 106 M−1. Addition of 30-S subunits to the 50-S subunits raised the number of molecules complexed by 50-S particles to 0.5 and increased KGTPa to 5.1 × 106 M−1, suggesting an important role of the 30-S subunit in the configuration of the nucleotide binding site. With purified endogenous polysomes, washed with either 1 M or 0.1 M NH4Cl, only 0.2–0.25 molecule of GTP was bound per ribosome at saturation and KGTPa was 6.7 × 106 M−1. Deacylation of nascent peptidyl chains raised these values to about 0.5 and 9.6 × 106 M−1, respectively. The small fraction of ribosomes that formed the complex in untreated polysomes appeared to be constituted mainly of true polysomal ribosomes, since (a) they possessed peptidyl-tRNA, as judged by the increased stability of their GDP · EF-G · ribosome · fusidic-acid complex in the presence of puromycin, and (b) they sedimented as polysomes. The results thus indicate the existence of a functional heterogeneity in the polysomal preparations.

Binding of putrescine and spermidine to ribosomes from Escherichia coli.

1 The binding of putrescine and spermidine to free 70-S ribosomes has been studied by equilibrium dialysis at 4°C in the presence of physiological concentrations of K+ and Mg2+. The number of binding sites for each polyamine is approximately 800, and the association constants for putrescine and spermidine are 80 and 500 mM−1, respectively. In the case of spermidine there is evidence that a small fraction of the sites (3–5% of the total) have an affinity for spermidine that is about 10 times higher. 2 Competition experiments suggest that the two polyamines bind at closely related sites on the ribosome. 3 The binding sites for spermidine are distributed in proportion between the two ribosomal subunits. 4 The S20, w of free 70-S ribosomes (62 S) is unaffected in the presence of physiological concentrations of the two polyamines. 5 The effect of pressure in promoting dissociation of 70-S ribosomes to subunits is reduced by putrescine and spermidine.

Silver plated ribosomes.

WE recently reported that dissociation of free 70S ribosomes from E. coli to 30S and 50S subunits could occur during sedimentation in buffer containing K+ as the predominant monovalent cation1. Spermidine at a physiological concentration (1 mM) specifically prevented the dissociation and we suggested that the dissociation that occurred in its absence was due to loss of the polyamine from the 70S ribosome. We have subsequently found that this hypothesis is wrong and that the dissociation that took place in the original experiments was due not primarily to loss of spermidine, but to a reaction with silver present in very small amounts in the AR KCl used in the buffer.

Stabilization of 70S ribosomes by spermidine.

RIBOSOMAL subunits in Escherichia coli dissociate and reasso-ciate after each round of translation1, but it is not known whether separation occurs at termination of protein synthesis2, or whether they are released as “free” 70S ribosomes and subsequently dissociate3. The existence of free 70S ribosomes in vivo is controversial because sedimentation analyses of cell extracts using K+ as the monovalent cation demonstrate significant numbers of 70S ribosomes, but when Na+ is used 30S and 50S subunits predominate4,5.

Amounts of free 70S ribosomes and ribosomal subunits found in [formula omitted] at various temperatures

When the rate of protein synthesis in cultures of Escherichia coli was decreased by lowering the temperature, free 70S ribosomes accumulated and the number of polysomes per cell decreased, while 50S and 30S ribosomal subunits remained almost constant.

Physiological role of 70S ribosomes in bacteria.

Evidence is presented indicating that free 70S ribosomes are real components of Bacillus stearothermophilus and Escherichia coli in exponential and stationary phases of growth. After pulses of radioactive leucine or uracil were given, protein synthesis was instantaneously stopped by chloramphenicol, or polypeptide growth was allowed to proceed to completion in slowly cooled cells. Analysis of label distribution in sucrose gradients showed that 70S monomers, and not subunits, are final products of translation.A ribosome cycle for protein synthesis in bacteria is proposed.