Comparison of cell-free protein synthesis by different regions of chicken brain

The cell-free protein synthesis by the postmitochondrial supernatant from chicken cerebrum was twofold greater than protein synthesis by the cerebellum or optic lobes. Ribosomal aggregation of mRNA and ribonuclease activity of the postmitochondrial supernatant from the three brain regions was not statistically different. The higher protein synthetic activity of the cerebral postmitochondrial supernatant was associated with both the postribosomal supernatant (cell sap) and microsomal fractions. Cerebral monomeric ribosomes were more active in polyuridylic acid directed polyphenylalanine synthesis than monomeric ribosomes from either the cerebellum or optic lobes. The ability of cerebral cell sap to support polyuridylic acid directed polyphenylalanine synthesis was 1.6 to 2 times greater than cell sap from the other two regions. Cell sap factors other than tRNA^phe or phenylalanyl-tRNA synthetases appear to be responsible for the higher protein synthetic activity of the cbr cell sap.     

In vitro Protein Synthesis by Polyribosomes of Peach Flower Buds at Different Physiological Stages

The in vitro phenylalanine incorporation by polyribosomes of peach flower buds (Prunus persica Stokes) during dormancy, dormancy break and flowering was investigated. Protein synthesis was measured using as catalyst either calf liver soluble factors or the ribosomal supernatant from the peach flower buds in the presence or the absence of the synthetic mRNA, polyuridylic acid. In the presence of polyuridylic acid, the activity of protein synthesis of dormant ribosomes is the same as that of ribosomes during dormancy break and flowering. The absence of synthetic messenger did not cause a change in activity. The ribosomal supernatant of dormant buds, but not of flowering buds, reduces the phenylalanine incorporation by polyribosomes from buds harvested at dormancy break.     

RIBOSOMAL ACTIVITY IN PRENATAL MOUSE BRAIN

Abstract— Regulation of protein synthesis is important for the proper growth and development of the brain. Our previous work on the regulation of protein synthetic activity in fetal mouse brain cell suspensions showed that the rate of protein synthesis decreased during the prenatal period. In the present study, ribosomal activity of cell-free homogenates and purified ribosomes obtained from fetal neural tissue was measured. The post-mitochondrial supernatant (PMS) fraction actively incorporated amino acids into polypeptides using either endogenous mRNA or polyuridylic acid as template. The protein synthetic activity was dependent upon the age of the fetus. Ribosomes purified from this fraction were also active in protein synthesis. Incorporation of phenylalanine was linear for 20 min, and dependent upon the concentration of ribosomes and the pH 5 enzyme fraction. The age dependent decrease in protein synthetic activity observed with the post-mitochondrial supernatant fractions was not found when these purified ribosomes were employed. Ribosomes obtained from fetal, newborn or adult neural tissue were compared and found equally active in their protein synthetic capacity.     

Regulation of Protein Synthesis in Zoospores of Blastocladiella

The factors responsible for the regulation of protein synthesis in the zoospores of Blastocladiella emersonii were studied by means of cell fractionation and in vitro assays. Charged transfer ribonucleic acid (tRNA) and aminoacyl-tRNA synthetases were found both inside the membrane-bound, ribosomal nuclear cap, and in the extracap cytoplasm. Ribosomes isolated from zoospore nuclear caps in low salt buffer failed to support polyuridylic acid-dependent phenylalanine incorporation. After washing with high salt buffer, the cap ribosomes were equivalent in activity to similarly prepared plant ribosomes. Both the high-salt wash from cap ribosomes and the extracap supernatant fraction contained an unidentified material which inhibited aminoacyl-tRNA binding and peptide bond formation by ribosomes. Ribosomal binding of polyuridylic acid was not inhibited. Washed cap ribosomes supported very low incorporation rates without added messenger RNA, and were highly dependent upon added poly U for phenylalanine incorporation, indicating a low level of messenger in nuclear caps. It is concluded that enclosure of the ribosomes in the nuclear cap does not in itself prevent protein synthesis, and that the lack of activity may be due to the presence of a ribosome inhibitor.     

Studies on the protein-synthesizing activity of the ribosomes of rat liver. The activity of free polysomes

1. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the presence and absence of polyuridylic acid. 2. The activity of a light-microsome fraction from adult liver was greater than that of a heavy-microsome fraction, and the light-microsome fraction was also more markedly stimulated by the presence of polyuridylic acid. 3. The light-microsome fraction, when analysed by density-gradient centrifugation, contained a higher ratio of free ribosomes to bound ribosomes, whereas the reverse was true for the heavy-microsome fraction. Similar results were obtained for liver from adult and 5-day-old rats. 4. When the light-microsome fraction was incubated under conditions in which amino acid was incorporated into protein there was only a small increase in the ratio of free to bound ribosomes. When such a fraction was incubated with [(14)C]leucine and was then subjected to density-gradient centrifugation the fraction with the highest specific activity based on RNA had a density between that of the bound and free ribosomes. Treatment of the incubated fraction with ribonuclease shifted the radioactivity towards the free ribosome peak. These properties are consistent with the presence of active free polysomes. Such a component appeared also to be present when the heavy-microsome fraction was incubated under similar conditions. 5. The effect of the presence of polyuridylic acid on the incorporation of [(14)C]phenylalanine by the light-microsome fractions from liver of adult and 5-day-old rats was greatest in the region of the free ribosomes, but it is probable that some small polysomes containing polyuridylic acid are formed. 6. Polyuridylic acid also stimulated the bound ribosomes to a small extent when the heavy-microsome fraction from the liver of young rats was incubated with [(14)C]phenylalanine. 7. The results are discussed in terms of the various morphological constituents in liver now known to play a role in the synthesis of protein for export and for the internal activity of the cell.     

Protein synthesis in aging soybean cotyledons. Loss in translational capacity

Ribosomes and supernatant fractions from soybean cotyledons of different ages were prepared to study the Poly U (polyuridylic acid)-directed phenylalanine incorporation. Ribosomes from younger cotyledons were more effective in phenylalanine incorporation compared to ribosomes from older cotyledons. Similarly, the supernatant fractions from younger cotyledons were more efficient, resulting in enhanced incorporation, than the older cotyledons. Substitution of wheat embryo supernatant fraction for soybean cotyledon supernatant fraction resulted in a several fold increase in amino acid incorporation activity, in ribosomes from all ages of soybean cotyledons. Such increase in activity was particularly significant in the older cotyledons. From these experiments it is concluded that in aging soybean cotyledons there is a loss in translational capacity.     

Localization of polyamine enhancement of protein synthesis to subcellular components of Escherichia coli and Pseudomonas sp. strain Kim.

At 5 mM Mg2+, spermidine stimulation of polyphenylalanine synthesis by cell-free extracts of Escherichia coli was found to be about 30 times greater than that by extracts of Pseudomonas sp. strain Kim, a unique organism which lacks detectable levels of spermidine. By means of reconstitution experiments, the target of spermidine stimulation was localized to the protein fraction of the highspeed supernatant component (S-100) of E. coli and was absent from, or deficient in, the S-100 fraction of Pseudomonas sp. strain Kim. The spermidine stimulation did not appear to be due to the presence in the E. coli S-100 fraction of ribosomal protein S1, elongation factors, or E. coli aminoacyl-tRNA synthetases. The failure to observe spermidine stimulation by the Pseudomonas sp. strain Kim S-100 fraction was also not due to a spermidine-enhanced polyuridylic acid degradation. The synthesis of polyphenylalanine by Pseudomonas sp. strain Kim extracts was stimulated by putrescine and by S-(+)-2-hydroxyputrescine to a greater degree than was synthesis by E. coli extracts. The enhancement by putrescine and by S-(+)-2-hydroxyputrescine with Pseudomonas sp. strain Kim extracts was found to be due to effects on its ribosomes.     

RIBOSOMAL SUBUNITS FROM NEONATAL MOUSE BRAIN HIGHLY ACTIVE IN POLYPHENYLALANINE SYNTHESIS

Abstract— A highly active subcellular protein synthesising system is described, in which uncomplexed ribosomes isolated from 5 to 7 day old mouse brain can be reprogrammed with polyuridylic acid. Either purified free polyribosomes or microsomes were used as the starting material for the preparation of uncomplexed ribosomes by treatment with 0.5 m -KCl and puromycin. After reduction of the salt concentration 80S ribosomes were isolated by washing through sucrose. When, subsequently, zonal centrifugation in equivolumetric sucrose gradients containing 0.5 m -KCI was performed, purified ribosomal subunits were obtained. Cross-contamination of subunits was less than 5%. Re-associated ribosomes and recombined isolated ribosomal subunits both showed high activities in vitro. Incorporation levels of 50–60 phenylalanine residues per ribosome could be reached, at a rate of 0.5–2.0 residues/min/ribosome, depending on the activity of the high speed supernatant enzymes added. It was shown by paper chromatography of the cell-free product that only oligophenylalanine formation takes place. It was estimated that 6&70% of the ribosomes present in vitro were actively participating in the protein synthesis process.     

Inhibitory Effect of Diphtheria Toxin on Amino Acid Incorporation in Escherichia coli Cell-Free System

The mechanism of action of diphtheria toxin in an Escherichia coli cell-free protein-synthesizing system was examined. When the washed ribosomes were incubated with toxin before addition of messenger ribonucleic acid (RNA), peptide syntheses of (14)C-phenylalanine directed by polyuridylic acid or phage R17 RNA were strongly inhibited by a small amount of toxin. Whereas, if the soluble fraction (105,000 x g supernatant fraction) was preincubated with toxin, no effect of toxin occurred either on the induced protein synthesis or on the activity of guanosine triphosphatase even in the presence of nicotinamide adenine dinucleotide. The binding of (3)H-formylmethionyl-transfer RNA to E. coli ribosomes directed by either R17 RNA or trinucleotide AUG was also decreased by toxin. These findings suggest that diphtheria toxin may prevent the binding of messenger RNA by successfully competing with the AUG for ribosomal binding sites. Sucrose-density gradient studies support this concept by showing the decrease in binding of (3)H-labeled R17 RNA to E. coli ribosomes exposed to toxin.     

Regulation of protein synthesis in heat-shocked Drosophila cells. Soluble factors control translation in vitro

We have developed an in vitro translation system from heat-shocked and normal Drosophila cultured cells. The lysates retain regulation of translation typical of the whole cells from which they were prepared, both when programmed by endogenous mRNA and when RNA-dependent. These systems have been used to investigate the mechanism of shutdown of normal protein synthesis and selection of heat shock mRNAs for translation in heat shock in Drosophila. Supplementation of intact RNA-dependent lysates with separated ribosome or supernatant fractions from normal or heat-shocked translation systems showed the normal supernatant fraction could "rescue" normal protein synthesis in a heat shock lysate. Normal ribosomes had no rescuing activity and neither heat shock fraction affected translation in normal lysates. Reconstitution of the system from separated ribosomes and supernatant in normal and mixed combinations showed heat shock and normal ribosomes were both competent to support normal protein synthesis with normal supernatant. Heat shock supernatant did not support normal protein synthesis with ribosomes from either source. We conclude that the factors regulating translation in heat-shocked Drosophila cells are soluble factors in the lysate and that the soluble factors present in the normal lysate are dominant.     

Protein synthesis by single ribosomes

The ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into an amino acid sequence. Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish the initiation, elongation, and termination of peptide synthesis. In translocating processively along the mRNA template during the elongation cycle, ribosomes act as supramolecular motors. Here we demonstrate that ribosomes adsorbed on a surface, as for mechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescent beads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individual ribosomes. This work opens the way for application of biophysical techniques, originally developed for the classical motor proteins, to the understanding of protein biosynthesis.     

Inhibition of protein synthesis by polypeptide antibiotics.. II. In vitro protein synthesis

Ennis, Herbert L. (St. Jude Children's Research Hospital, Memphis, Tenn.). Inhibition of protein synthesis by polypeptide antibiotics. II. In vitro protein synthesis. J. Bacteriol. 90:1109-1119. 1965.-This investigation has shown that the polypeptide antibiotics of the PA 114, vernamycin, and streptogramin complexes are potent inhibitors of the synthetic polynucleotide-stimulated incorporation of amino acids into hot trichloroacetic acid-insoluble peptide. The antibiotics inhibited the transfer of amino acid from aminoacyl-soluble ribonucleic acid (s-RNA) to peptide. The A component of the antibiotic complex was active alone in inhibiting in vitro protein synthesis, whereas the B fraction was totally inactive. However, the A component, when in combination with the B component, gave a greater degree of inhibition than that observed with the A fraction alone. On the other hand, the endogenous incorporation of amino acid was much less susceptible to inhibition than the incorporation of the corresponding amino acid in a system stimulated by synthetic polynucleotide. In addition, synthesis of polyphenylalanine stimulated by polyuridylic acid was inhibited to a greater extent when the antibiotics were added before the addition of polyuridylic acid to the reaction mixture than when the antibiotics were added after the polynucleotide had a chance to attach to the ribosomes. However, the antibiotics apparently did not inhibit the binding of C(14)-polyuridylic acid or C(14)-phenylalanyl-s-RNA to ribosomes. The antibiotics did not affect the normal release of nascent protein from ribosomes and did not disturb protein synthesis by causing misreading of the genetic code. The antibiotics bind irreversibly to the ribosome, or destroy the functional identity of the ribosome. The antibiotic action is apparently a result of the competition between antibiotic and messenger RNA for a functional site(s) on the ribosome.     

Tetracycline inhibition of cell-free protein synthesis. II. Effect of the binding of tetracycline to the components of the system

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. II. Effect of the binding of tetracycline to the components of the system. J. Bacteriol. 92:197-203. 1966.-When tetracycline, an inhibitor of cell-free protein synthesis, was preincubated with each component of the Escherichia coli cell-free system, i.e., ribosomes, soluble ribonucleic acid (sRNA), polyuridylic acid (poly U), and S-100 (supernatant enzymes), only the ribosomal-bound antibiotic was inhibitory to the cell-free assay. Experiments designed to further localize the site of inhibition to either the 50S (Svedberg) or the 30S ribosomal subunit were not conclusive. Tritiated tetracycline (7-H(3)-tetracycline) was bound to isolated 50S ribosomes, and these were recombined with 30S subunits to form 70S ribosomes. When these ribosomes were dissociated and the subunits reisolated, the antibiotic was found with both the 50S and the 30S particles. The same results were observed when the tetracycline was initially bound to the 30S subunit.     

CYTOPLASMIC SYNTHESIS OF NUCLEAR PROTEINS : Kinetics of Accumulation of Radioactive Proteins in Various Cell Fractions after Brief Pulses

The synthesis of cytoplasmic and nuclear proteins has been studied in HeLa cells by examining the amount of radioactive protein appearing in the various subcellular fractions after labeling for brief periods. Due to the rapid equilibration of the amino acid pool, the total radioactivity in cytoplasmic protein increases linearly. The radioactivity observed in the cytoplasm is the sum of two components, the nascent proteins on the ribosomes and the completed proteins. At very short labeling times the specific activity of newly formed proteins found in the soluble supernatant fraction (completed protein) increases as the square of time, whereas the specific activity of the ribosomal fraction (nascent protein) reaches a plateau after 100 sec. The kinetics of accumulation of radioactive protein in the nucleus and the nucleolus is very similar to that of completed cytoplasmic protein, which suggests that the proteins are of similar origin. The rate of release and migration of proteins from the ribosomes into the nucleus requires less time than the synthesis of a polypeptide, which is about 80 sec. The uptake of label into nucleolar proteins is as rapid as the uptake of label into proteins of the soluble fraction of the cytoplasm, while nuclear proteins, including histones, tend to be labeled more slowly. The same results are obtained if protein synthesis is slowed with low concentrations of cycloheximide. The kinetics of incorporation of amino acids into various fractions of the cell indicates that the nucleus and the nucleolus contain few if any growing polypeptide chains, and thus do not synthesize their own proteins.     

Protein Synthesis in a Cell-Free Extract from Staphylococcus aureus

Cell-free Staphylococcus aureus extracts have been prepared which actively incorporate amino acids into protein. The requirements for amino acid incorporation of this preparation were strongly suggestive of de novo protein synthesis, since it showed an absolute requirement for ribosomes, 105,000 × g supernatant fluid, energy source, and magnesium ion. The stability of these extracts was greatly improved by use of dithiothreitol instead of mercaptoethanol as a sulfhydryl protecting reagent. Data were presented to show that the binding of aminoacyl-soluble ribonucleic acid to ribosomes did not require guanosine triphosphate and supernatant enzyme. The major characteristic which distinguishes this system from other cell-free systems is the much higher magnesium concentration required to maintain ribosomes intact and to obtain the maximal incorporation of amino acids. Addition of polyuridylic acid, polyadenylic acid, or polycytidylic acid caused about 60-fold, 30-fold, or 4-fold stimulation of the incorporation of phenylalanine, lysine, or proline, respectively. Studies by density gradient sedimentation indicated that radioactive polyuridylic acid or polyadenylic acid was associated with the monosomes. This complex can actively synthesize polypeptides. On the other hand, the nascent protein synthesized under the direction of endogenous messenger ribonucleic acid was associated with both polysomes and monosomes.     

Vicia sativa L. 'Run-off' and Purified Ribosomes: Polyphenylalanine Synthesis and Molecular Action of Ribosome-inactivating Proteins

A ‘run-off’ cell-free translation system (a 30000x g supernatant; S 30) has been prepared from 4–5-d-oldembryonic axes of Vicia sativa L., a plant lacking ribosome-inactivatingprotein activities which is very sensitive to certain RIPs butnot to others. The system was able to generate a high rate ofpolyphenylalanine synthesis upon addition of polyuridylic acid.From this supernatant, purified ribosomes and a 100000 x g supernatantwere prepared which were able to perform polyphenylalanine synthesiswhen mixed together (reconstituted system). The most importanttranslation parameters were optimized in each case. Both theS 30 and the reconstituted system displayed differential sensitivitiesto certain RIPs. The purified RIP-inactivated ribosomes wereable to release a 370 nucleotide rRNA fragment diagnostic forRIPs upon treatment of the isolated rRNA with acid aniline. Key words: Translation, polyphenylalanine synthesis, ribosomes, ribosome-inactivating proteins, Vicia sativa     

Functional modifications of the translational system in Bacillus subtilis during sporulation.

Extracts of sporulating cells were found to be defective in vitro translation of phage SP01 ribonucleic acid (RNA) and vegetative Bacillus subtilis RNA. The activity of washed ribosomes from sporulating cells was very similar to that of washed ribosomes from vegetative cells in translating polyuridylic acid, SP01 RNA, and vegetative RNA. The S-150 fraction from either vegetative or sporulating cells grown in Difco sporulation medium contained an apparent inhibitor of protein synthesis. The crude initiation factor fraction from ribosomes of sporulating cells was defective in promoting the initiation factor-dependent translation of SP01 RNA. The crude initiation factor preparations from sporulating cells were as active as the corresponding preparations from vegetative cells in promoting the initiation factor-dependent translation of either phage Qbeta or phage T4 RNA by washed Escherichia coli ribosomes. The crude initiation factors from sporulating cells were perhaps more active than those from vegetative cells in promoting the initiation factor-dependent synthesis of phage T4 lysozyme by E. coli ribosomes. The crude initiation factor preparations from either vegetative or stationary-phase cells of an asporogenous mutant showed similar ability to promote the in vitro translation of SP01 RNA.     

The production of biologically active subparticles from rabbit reticulocyte ribosomes

THE EFFECT OF EXPOSING RABBIT RETICULOCYTE RIBOSOMES TO CONCENTRATED SOLUTIONS OF POTASSIUM CHLORIDE WAS EXAMINED BY: (a) dialysis against 0.5m-potassium chloride; (b) zone centrifugation through a sucrose gradient in 0.5m-potassium chloride; (c) differential centrifugation of a solution made 0.5m with respect to potassium chloride. The products of each treatment and their ability to support protein synthesis in a reticulocyte cell-free system, in the presence and in the absence of polyuridylic acid, were examined. The following results were found. (1) Exposing the polysomes to 0.5m-potassium chloride was not a sufficient condition for the complete dissociation of ribosomes into subparticles; the reaction showed a concentration-dependence, implying the existence of an equilibrium between the various ribosomal species. Disturbance of the equilibrium by removing certain products, as in zone centrifuging, can lead to complete dissociation. (2) The subparticles produced by dialysis or sucrose-gradient fractionation were biologically inactive and unstable. (3) The pellet obtained by differential centrifuging consisted of subparticles, if suspended in a Mg(2+)-free buffer; addition of Mg(2+) converted about 30% of the material into heavier sedimenting species, and the preparation had 20-40% of the activity of the untreated control polysomes in the cell-free system. Addition of the 0.5m-potassium chloride supernatant fraction resulted in further apparent reconstitution of sub-particles into ribosomes and polysomes and in a 50-100% restoration of biological activity. When both polyuridylic acid and supernatant factors were present incorporations similar to or higher than those of the control were attained.     

The effect of age on protein synthesis in mouse liver

1. A system of microsomes and 105000g supernatant from livers of old mice is less able to promote the incorporation of [(14)C]phenylalanine into protein than a similar system from livers of young animals. 2. The decrease in [(14)C]phenylalanine incorporation is attributable to changes in microsomes from old animals rather than in the cell-sap fraction. 3. Decreased synthetic ability is found in various classes of microsomes from older animals, namely unfractionated, light and heavy microsomes, but not in detergent-washed ribonucleoprotein particles. 4. Deletions of certain detergent-soluble microsomal proteins accompany the decreased synthetic ability of microsomes from older animals. 5. Microsomes from old mice are less responsive to a synthetic messenger RNA, polyuridylic acid, and this is partly due to a higher rate of hydrolysis in the presence of cell sap from animals of extreme age. 6. Other more direct evidence, from the priming of a cell-free protein-synthesizing system from bacteria and the examination of ribonucleoprotein particles on sucrose density gradients, suggests that senescence is accompanied by a decrease in messenger RNA content.     

Regulation of Prenatal and Postnatal Protein Synthesis in Mouse Brain

Abstract: Regulation of protein synthesis during prenatal and postnatal brain development was examined using postmitochondrial supernatant (PMS) fractions and isolated ribosome-pH 5 enzyme systems from fetal, neonatal, and adult neural tissue. The rate of polyuridylic acid (poly-U)-dependent protein synthetic activity was inversely proportional to the endogenous rate of protein synthesis in either the PMS fractions or ribosomal preparations. A careful analysis of the kinetics of the poly-U-dependent polypeptide synthesis revealed that there was a lag in the time at which certain of the PMS preparations could begin to utilize the poly-U template as sole source of mRNA. The lag period was dependent upon the developmental age of the neural tissue used and the Mg²⁺ concentration of the protein synthesis reaction. Since previous work reported that the observed developmental decrease in the rate of polypeptide synthesis utilizing a poly-U template could not be measured in a purified ribosomal-pH 5 enzyme system, ribosomes were obtained by several isolation techniques to determine if the purification procedure might have affected the ribosomes in some manner by removing a specific protein(s) involved in ribosome-cytosol interactions. At 6 mM-Mg²⁺ the rate of poly-U-dependent protein synthesis was inversely proportional to the rate of endogenous synthesis and depended upon the method used to isolate the ribosomes: microsomes ∼Triton X-100-treated < DOC-treated < KCl-treated. However, there was no age-dependent effect with any of the ribosomal preparations. The data suggest that there is a developmental modulating effect of ribosomal activity in PMS preparations which is not found in association with the isolated ribosome-pH 5 enzyme protein synthesizing system.