Isolation of active 30S and 50S ribosomal subunits from a psychrotrophic bacterium

Ribosomes from the psychrotroph,Bacillus insolitus, were successfully dissociated into 30S and 50S ribosomal subunits, which were active in carrying out in vitro protein synthesis, measured by the poly U-directed incorporation of¹⁴C-l-phenylalanine into polyphenylalanine. As opposed to the undissociated ribosomes, which are heat sensitive (30°C), the dissociated ribosomes were not thermally sensitive. The heat-sensitive component(s) was found to be removed from the ribosomes during dissociation into 30S and 50S ribosomal subunits; when added back to the ribosomal subunits, heat sensitivity was conferred.     

Adaptive Potential of Wheat Ribosomes toward Heat Depends on the Large Ribosomal Subunit and Ribosomal Protein Phosphorylation

In a study of the translational efficiency of ribosomal subunits as a function of an in vivo temperature pretreatment, ribosomes were isolated from heat-pretreated (36°C) and reference (20°C) wheat seedlings (Triticum aestivum L.). The efficiency of recombined subunits in translating polyuridylic acid was assessed. A threefold increase in the rate of incorporation of phenylalanine by ribosomes from heat-pretreated plants was due to the large ribosomal subunit. This adaptive temperature effect was not correlated with a higher thermal stability of ribosomes or subunits from heat-pretreated seedlings, and two-dimensional gel electrophoresis failed to detect structural alterations of ribosomal proteins. Phosphorylation of ribosomal proteins in vitro showed no differences between ribosomes or subunits from heat-pretreated and reference plants. Incubation with [³²P]orthophosphate in vivo led to twice the amount of phosphate in ribosomal proteins from heat-pretreated wheat seedlings. This result is important with respect to the evaluation of the molecular basis of enhanced translational efficiency of ribosomes isolated from heat-pretreated wheat seedlings.     

Studies on the interaction between ribosomes and 14 CH 3 -F 3 initation factor

Summary Initiation factor F₃ has been purified fromEscherichia coli and labelledin vitro by reductive alkylation. The¹⁴CH₃–F₃ so obtained had a specific activity of about 1 000 cpm/g and was shown to have retained its biological activity. Labelled F₃ binds to 30S ribosomal subunits ofEscherichia coli andBacillus stearothermophilus, but does not bind to either 70S ribosomes or 50S ribosomal subunits. The stoichiometry of the binding indicates that one molecule of¹⁴CH₃–F₃ is bound to each 30S ribosomal subunit. Several antibiotics, known to interact with 30S subunits, inhibit the binding. Functional studies indicate that F₃ is released from 30S ribosomes as a result of the formation of the 70S initiation complex.     

Metabolism ofAedes aegypti cells grown in vitro. 1. Incorporation of3H-uridine and14C-leucine

Summary Metabolic activity ofA. aegypti cells grown in vitro has been studied by incorporation of³H-uridine and¹⁴C-leucine. “Chase” experiments with unlabeled precursors, and the use of actinomycin D and puromycin, showed that³H-uridine was incorporated into cellular RNA, and that¹⁴C-leucine was incorporated into protein of these cells. Incorporation of³H-uridine was inhibited when actinomycin D was used at a concentration of 10 μg/ml, and¹⁴C-leucine incorporation was inhibited to the same extent by puromycin at a concentration of 100 μg/ml medium. Contribution No. 148.     

Inhibition of the incorporation of14C-precursors inMycobacterium smegmatis by antibiotics and chemotherapeutics

Twenty antituberculostatics and twelve other compounds were divided into three groups according to their ability to influence the rate of incorporation of¹⁴C-adenine and¹⁴C-leucine inM. smegmatis. The first group includes compounds significantly inhibiting the incorporation of¹⁴C-leucine, the second group comprises compounds inhibiting simultaneously the incorporation of both¹⁴C-precursors, the third group contains compounds that do not bring about a 50% decrease of the rate of incorporation even at a concentration of 400 μg/mL.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Developmental changes in gene expression during pollen differentiation and maturation inNicotiana tabacum L

Total and polysome-bound ribosomes and the uptake and incorporation of³H-uridine and¹⁴C-leucine were examined in dividing microspores and in pollen grains isolated from anthers of 6 different developmental stages. Direct evidence was obtained that the formation of cytoplasm of the vegetative cell following microspore division is related to a rapid activation of RNA and protein synthesis and of ribosomes in differentiating pollen. Total ribosomes associated with gametophytic programme rose about 10times and the process of differentiation was accompanied by a rapid increase in uptake capacity of pollen grains for both uridine and leucine. Pollen development after cytoplasm synthesis and starch deposition continued by pollen maturation, which was characterized by a decline in RNA synthesis, dissociation of polysomes and by a further rise of transport activity of pollen grain wall for exogenous substrates, indicating probable pollen adaptation for utilization of metabolites from the degenerating tapetal cytoplasm.     

The distribution of ribosomes between different functional states in livers of fed and starved mice

1. To investigate the role of ribosome function in regulating protein synthesis, the activity, distribution and functional states of ribosomal particles were investigated in livers of mice fed ad libitum or starved overnight. 2. The distribution of protein-synthesizing activity between polyribosomes of different sizes was analysed after incorporation of radioactive leucine, and the quantitative distribution of ribosomes as native subunits, monomers and polyribosomes was analysed after incorporation of orotic acid. Precursors labelled with ³H or ¹⁴C were given separately to fed and starved mice, so that livers from the two groups of animals were processed together. 3. The former experiments showed that starvation has little effect on the distribution of protein-synthesizing activity across polyribosome sedimentation patterns, though the latter experiments showed that the proportion of ribosomes existing as monomers increased from 9.5% to 15.2%, whereas the proportion existing as polyribosomes decreased from 81.4% to 75.6%. Starvation had a negligible effect on the proportion of native subunits, which accounted for 9.1% and 9.2% of the ribosomes in fed and starved mice respectively. 4. The monomeric ribosome fraction was isolated and subjected to ionic conditions which selectively dissociate single ribosomes. Starvation increased the proportion of monomers that dissociated from 59% to 72%, so the monomers that accumulate in livers of starved animals are single ribosomes and not monoribosomes resulting from degradation of polyribosomes. 5. The fate of newly formed ribosomal particles was studied by measuring the specific radioactivity of native subunits, monomers and polyribosomes at different times after injection of radioactively labelled orotic acid. Starvation did not appear to affect equilibration between newly formed particles and polyribosomes, and the radioactivity of polyribosomes in both groups of mice reached about 90% of that in native subunits after 4h. The radioactive labelling of monomers proceeded at a slower rate, especially after starvation. At 4h, the radioactivity of monomers was 64% and 55% that of native subunits in fed and starved mice respectively.     

Synthesis and biological activity of [14a-3H]cryptopleurine

[14a-³H]Cryptopleurine was chemically synthesized from the perchlorate salt of 9,11,12,13,14,15-hexahydro-2,3,6-trimethoxyphenanthro(9,10-b)quinolizidinium by reduction with NaB³H₄. The [³H]cryptopleurine was recrystallized from acetone and further purified by chromatography through alumina using benzene as the eluting solvent. Both infrared and ultraviolet spectra of the labeled product were identical to those obtained using either the natural compound or the unlabeled synthetic compound. Thin-layer analysis on various solid supports using several different eluting solvents gave only one radioactive spot with a specific activity of 1438 Ci/mol, which in all cases cochromatographed with the natural sample. The [³H]cryptopleurine was also identical to the unlabeled compound in that it bound strongly to polyribosomes. 80 S ribosomes, and 40 S ribosomal subunits, all isolated from yeast. Binding was less strong using either 60 S ribosomal subunits or Escherichia coli ribosomes.     

Characterization of ribosomes of a strain ofStreptomyces aureofaciens producing chlortetracycline

These studies were designated to investigate the effect of chlortetracycline on sedimentation properties of polysomes and ribosomes present in the chlortetracycline producing strain ofStreptomyces aureofaciens. In presence of chlortetracycline polysomes and ribosomes are more stable than the bacterial ones. At lower chlortetracycline concentrations (1–5 μg/ml) dissociation of polysomes into 70 S monomers was not observed. Ribosomes in higher concentration of chlortetracycline (400 μg/ml) form aggregates. A decrease of Mg²⁺ to 0.1mm caused dissociation of ribosomes to two subunits and in this state none of indicated concentrations of chlortetracycline caused aggregation. The exact sedimentation values of ribosomes and ribosomal subunits were calculated from extrapolation to infinite dilution. S_20,w for monomer form was 68.8, and for ribosomal subunits 49.8 and 31.2 respectively. Ribosomal RNA sedimentates as two Schlieren peaks of 16 S and 22 S. It was found that 30 S subunits contain 15 structural proteins, while 21 proteins were resolved from 50 S subunits.     

Effects of Ammonium and Potassium Ions on Some Physiological and Biochemical Processes of Excised Cucumber Cotyledons

Incubation of excised cucumber cotyledons (Cucumis sativus L.) with NH₄Cl solutions exceeding 0.001 M inhibited their greening, fresh weight increases, and incorporation of ¹⁴C-leucine into insoluble N compounds. The absorption of ¹⁴C-leucine during incubation and retention of moisture by the excised cotyledons after incubation were greatly diminished by the NH₄Cl treatments. Treatment with KCl solutions of the same concentrations as the NH₄Cl stimulated the greening, fresh weight increases, and the absorption and incorporation of ¹⁴C-leucine. Desiccation of cotyledons stored at 5°C for 10 days was inhibited by dilute KCl solutions. The toxicity of NH₄Cl was alleviated by KCl solutions at low concentrations.     

ISOLATION OF CYTOPLASMIC AND CHLOROPLAST RIBOSOMES AND THEIR DISSOCIATION INTO ACTIVE SUBUNITS FROM CHLAMYDOMONAS REINHARDTII

A mixture of cytoplasmic (80S) and chloroplast (70S) ribosomes from Chlamydomonas reinhardtii was freed of contaminating membranes by sedimentation of the postmitochondrial supernatant through a layer of 1.87 M sucrose. The purified ribosomes were separated into 80S and 70S fractions by centrifugation at a relatively low speed on a 10–40% sucrose gradient containing 25 mM KCl and 5 mM MgCl₂. Both the 80S and 70S ribosomes were dissociated into compact subunits by centrifugations in 5–20% high-salt sucrose gradients. The dissociations of both ribosomal species under these conditions were not affected by the addition of puromycin, indicating that the ribosomes as isolated were devoid of nascent chains. Subunits derived from the 80S ribosomes had apparent sedimentation coefficients of 57S and 37S whereas those from the 70S ribosomes had apparent sedimentation coefficients of 50S and 33S. In the presence of polyuridylic acid and cofactors, the 80S and 70S ribosomes incorporated [¹⁴C]phenylalanine into material insoluble in hot TCA. The requirements for incorporation were found to be similar to those described for eukaryotic and prokaryotic ribosomes. Experiments with antibiotics showed that the activity of the 80S ribosomes was sensitive to cycloheximide, whereas that of the 70S ribosomes was inhibited by streptomycin. The isolated subunits, when mixed together in an incorporation medium, were also active in the polymerization of phenylalanine in vitro.     

The mechanism of action of initiaton factor 3 in protein synthesis,I. Studies on ribosomes crosslinked with dimethylsuberimidate

The mechanism of action of chain initiation factor 3 in translation was examined by using $\text{E. coli}$ 70S ribosomes which were covalently crosslinked with dimethylsuberimidate. Crosslinked ribosomes were inactive in AUG-dependent fMet-tRNA binding, and were not stimulated by IF-3 in poly(U) translation. IF-3 is known to be required for maximal rates of amino acid incorporation with synthetic polynucleotides at 18 mM Mg²⁺. A direct interaction of IF-3 with 70S ribosomes was demonstrated by crosslinking ¹⁴C-labeled IF-3 to 70S ribosomes. The labeled factor was also crosslinked to 30S and 50S ribosomal subunits. A model is presented proposing the mechanism of action of IF-3 on 70S ribosomes.     

Energetic aspects of spore germination in filamentous fungi

The antifungal activity of substances interfering with the function and biogenesis of mitochondria was studied. Strict anaerobiosis, cyanide, azide, oligomycin, bongkrekic acid and ethidium bromide were found to prevent spore germination ofAspergillus niger andPenicillium italicum in liquid germination medium. The effect of azide, oligomycin and ethidium bromide was fungicidal. Cyanide and azide completely inhibited the incorporation of¹⁴C-leucine and¹⁴C-uracil into germinating conidia ofA. niger. Oligomycin and ethidium bromide reduced the extent of incorporation of both precursors in the first few hours of conidial germination and at later stages stopped it completely. The inhibition of both spore germination and macromolecules synthesis during the germination ofA. niger conidia were in relation to the specific inhibitory effect of the agents on respiratory activity of dormant conidia and mycelial cells. The results indicate that both the function of mitochondrial genetic and protein synthesizing systems and the function of oxidative phosphorylation are essential for normal spore germination and fungal growth.     

Changes in ribosomal activity during incubation of Daucus carota root disks

Cytoplasmic monoribosomes from freshly cut and ‘aged’ carrot root disks were characterized relative to the Mg²⁺ optima for poly U (polyuridylic acid)-directed phenylalanine incorporation, the ease of dissociation by KCl in the presence of Mg²⁺, the ability to bind ³H-poly U, and acrylamide gel fractionation of the ribosomal proteins. The differences in in vitro amino acid incorporation by ribosomes and supernatant from fresh and ‘aged’ disks were confined to the ribosome fraction. The Mg²⁺ optima for poly U-directed ¹⁴C-phenylalanine incorporation was 16 mM for ribosomes from ‘aged’ disks compared to 20 mM for ribosomes from fresh disks. Monoribosomes from the fresh disks were easily dissociated into subunits (0·2 M KCl in 5 mM Mg²⁺) while the ribosomes from ‘aged’ disks were not completely dissociated even in 0·5 M KCl. Ribosomes from ‘aged’ disks were more effective in binding ³H-poly U than ribosomes from fresh disks. When the disks were subjected to an anaerobic environment prior to ribosome extraction (to strip monoribosomes of peptidyl-t RNA) the above effects of ‘aging’ were reversed. These results suggest that increased monoribosome activity associated with ‘aging’ may be related in part to an increase in the level of peptidyl-tRNA associated with the ribosomes. Acrylamide gel electrophoresis profiles of ribosomal proteins extracted from ribosomes of fresh and ‘aged’ tissue suggest that a change in the protein complement may also be important to the observed changes in ribosomal activity. The ribosomes from ‘aged’ disks contained at least two components not associated with ribosomes from fresh disks.     

Functional Inactivation and Appearance of Breaks in RNA Chains Caused by Gamma Irradiation of Escherichia coli Ribosomes

70S ribosomes and 30S ribosomal subunits from Escherichia coli MRE 600 were exposed to gamma irradiation at -80szC. Exponential decline of activity with dose was observed when the ability of ribosomes to support the synthesis of polyphenylalanine was assayed. Irradiated ribosomes showed also an increased thermal lability. D₃₇ values of 2.2 MR and 4.8 MR, corresponding to radiation-sensitive molecular weights of 3.1 × 10⁵ and 1.4 × 10⁵, were determined for inactivation of 70S ribosomes and 30S subunits, respectively. Zone sedimentation analysis of RNA isolated from irradiated bacteria or 30S ribosomal subunits showed that at average, one chain scission occurs per four hits into ribosomal RNA. From these results it was concluded that the integrity of only a part of ribosomal proteins (the sum of their molecular weights not exceeding 1.4 × 10⁵) could be essential for the function of the 30S subunit in the polymerization of phenylalanine. This amount is smaller if the breaks in the RNA chain inactivate the ribosome.     

Amino Acid incorporation in developing fucus embryos

The incorporation of ¹⁴C-leucine and ¹⁴C-amino acid mixture into protein in unfertilized eggs and developing embryos of the brown alga Fucus vesiculosus L. was studied. Bacterial contamination was initially a problem, but it was found that the addition of 40 μg/ml chloramphenicol to the incubation medium would inhibit bacterial protein synthesis without affecting early development of the Fucus embryos. The kinetics of uptake and incorporation of ¹⁴C-leucine into the trichloroacetic acid-soluble and -insoluble fractions indicated that the exogenous precursor did not equilibrate with the main soluble leucine pool before incorporation into protein. Uptake and incorporation of leucine by embryos 90 to 175 minutes old were proportional to exogenous leucine concentration over the range 5 × 10⁻⁶ m to 5 × 10⁻³ m. Unfertilized eggs will incorporate ¹⁴C-leucine into protein. The rate of this incorporation increases dramatically in newly fertilized eggs with a maximum rate at 3.5 hours, a period of cell wall formation and increasing metabolic rates. Thereafter, the rate of incorporation declines until approximately 15 to 17 hours when it increases again concurrently with the onset of rhizoid initiation and cell division.     

Amino acid incorporation by nuclear membrane fraction of rat liver.

Nuclear membrane fraction of rat liver is able to incorporate ¹⁴C-leucine into its proteins $\text{in vitro}$. The incorporation of ¹⁴C-leucine into the nuclear membrane fraction was almost completely inhibited by chloramphenicol, but the inhibition by cycloheximide and puromycin was not so remarkable. RNase and DNase were not effective. The incorporation was also inhibited by several reagents known to interfere with energy metabolism. These characteristics of the incorporation of ¹⁴C-leucine by the nuclear membrane fraction are quite similar to those of the incorporation by nuclei isolated from rat liver and mitochondrial fraction, but seem to be different from those of the ordinary protein synthetic system in microsomal fraction. ¹⁴C-Leucine was preferentially incorporated into intrapolypeptide or C-terminal residues but not into N-terminal residues. Acrylamide gel electrophoresis showed that three protein species were mainly labelled. The incorporating activity of the nuclear membrane fraction obtained from regenerating liver 17 h after partial hepatectomy showed 220 % of the control. The possibility that the contaminated mitochondrial fraction might be responsible for the incorporation of ¹⁴C-leucine by the nuclear membrane fraction was ruled out.