Spermidine Requirement for Bacillus thuringiensis Ribosomes in Cell-Free Phenylalanine Incorporation

A cell-free system from Bacillus thuringiensis was found to actively incorporate phenylalanine into hot trichloroacetic acid-precipitable material in the presence of synthetic polynucleotide, ribosomes, S-100 supernatant, an energy-generating system, and guanosine triphosphate. Phenylalanine incorporation was absolutely dependent on the presence of spermidine in addition to magnesium ions, even when highly purified ribosomes were used. The spermidine effect could not be attributed to inhibition of nucleases. The ribosomal and supernatant fractions from Escherichia coli and B. thuringiensis could be substituted for each other in this system. The spermidine requirement was shown to be limited to the B. thuringiensis ribosome fraction.     

Protein synthesizing systems from spores and vegetative cells of Bacillus cereus

A system of polyphenylalanine synthesis was optimized for a comparison of the polymerizing activities of ribosomes from spores and vegetative cells of Bacillus cereus T. Ribosomes of both types react similarly, showing a magnesium optimum of about 6 mM and spermidine optima of about 5 mM and 4 mM for vegetative and spore ribosomes, respectively. These lead to optimum mono- to multivalent cation rations of 9 and 10 respectively at 100 mM ammonium ion. A comparison of the response of these ribosomes to suboptimal concentrations of magnesium and spermidine show that they differ qualitatively from each other, suggesting that they possess different structure, macromolecular or ionic components.Abbreviations DFP diisopropylfluorophosphate - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid     

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.     

Hemoglobin transition in erythrocytes of developing chick. Studies with cell-free protein-synthesizing systems.

Cell-free hemoglobin-synthesizing systems from erythrocytes of 4- and 17-day chick embryos have been developed. These systems have been used to investigate possible structural and functional differences in factors involved in protein synthesis obtained from these different developmental stages. Each cell-free system consists of three major cellular fractions i.e., the S-100 supernatant, the salt-washed ribosomes, and the 0.5 m KCl ribosomal wash. When the ribosomal wash fraction from one developmental stage is included in a cell-free system containing ribosomes and S-100 supernatant from the other developmental stage, a drastic reduction in the kinetics of [³H]leucine incorporation into globin products is observed, when compared to the homologous control cell-free systems. A similar depression of the kinetics of incorporation is observed when the mixed component is either the S-100 supernatant or the ribosomes. Control rates of incorporation can be reestablished when the corresponding homologous component is added back to the incubation mixture. The predominant types of hemoglobins produced in the salt-wash heterologous systems are those hemoglobins characteristic of the developmental stage of the salt wash. This seems to imply that the ribosomal salt-wash fraction may possess developmental stage specificity for the globins.     

Cell-Free Protein Synthesis by Rumen Protozoa

SYNOPSIS. The characteristics of protein synthesis by cell-free extracts of mixed rumen protozoa have been investigated. ATP,¹ GTP, and an energy supply system were necessary for amino acid incorporation which was partially inhibited by cycloheximide but not by chloramphenicol (100 μg/ml). The system was particularly sensitive to the cation concentration of the incubation mixture, maximal incorporation requiring 5 mM Mg⁺⁺ and 50 mM K⁺ Incorporation was further stimulated by the addition of 0.25 mM spermidine or 0.25 mM MnCl₂. Sucrose gradient centrifugation of the cell sap after amino add incorporation showed that most of the incorporated radioactivity was associated with free polysomes. These polysomes contained 82 S ribosomes which dissociated in high Tris concentrations to yield 40 S and 55 S ribosomes.     

Optimal isolation conditions for rainbow trout (Salmo gairdneri) hepatic ribosomes

1. Isolation conditions for rainbow trout (Salmo gairdneri) liver ribosomes were optimized. 2. Optimal initial buffer (Buffer I) concentrations were 250 mM sucrose, 50 mM Tris (pH 7.6, 25 degrees C), 75 mM KCl, and 5 mM MgSO4.7H2O. 3. Optimal concentrations for post-105 supernatant buffer (Buffer III) were 25 mM Tris (pH 7.6, 25 degrees C), 75 mM KCl, and 8 mM MgSO4.7H2O.     

Protein synthesis and the viability of rye grains. Loss of activity of protein-synthesizing systems in vitro associated with a loss of viability

A study was made of the integrity of some components of the protein-synthesizing system from viable and non-viable embryos of rye grains. In comparison with viable-embryo components both post-ribosomal supernatant and ribosomal fractions from non-viable embryos are impaired, for neither will fully support polyphenylalanine synthesis in poly(U)-directed cell-free systems. The lesion in the supernatant lies in components other than the tRNA or the aminoacyl-tRNA synthetase, for these are as functional as those present in the fully active cell-free systems from viable embryos. The ribosomes of embryos of lowered viability show considerable fragmentation and degradation of both 18S and 25S rRNA. This breakdown does not, however, account for the complete lack of polypeptide synthesis in the poly(U)-directed non-viable-embryo system, for if provided with viable-embryo supernatant, non-viable-embryo ribosomes will sustain 60% of the viable-embryo ribosome activity. A lesion in non-viable-embryo supernatant has been located in the binding of the aminoacyl-tRNA to the ribosome. The impaired components in both supernatant and ribosomes in systems in vitro may reflect the site of faults in protein synthesis in vivo in the early hours of germination. The development of these lesions during grain storage could contribute to senescence and loss of viability in the embryos of rye.     

Antagonistic action between spermidine and putrescine on association and dissociation of purified, run-off ribosomes from Escherichia coli.

The effects of polyamines on the equilibrium between prokaryotic ribosomal subunits and 70 S ribosomes have been studied as a function of concentration of Mg2+ from 2.5 to 7.5 mM. Run-off ribosomes were obtained from Escherichia coli and were washed with buffered 1 M NH4C1. Spermidine at 1 mm favors association of subunits at all concentrations of Mg2+. Putrescine, at concentrations above 8 mM, favors net dissociation at concentrations of Mg2+ below 4.5 mM. Streptomycin behaves like spermidine, while putrescine behaves like initiation factor 1 and initiation factor 3. The effect of putrescine on dissociation is time-dependent and appears to have a half-life of about 3.5 min at 30 degrees. When added after the effects of spermidine or streptomycin on association have occurred, putrescine still causes dissociation. The data suggests that putrescine may reduce net formation of vacant 70 S ribosomes. Another possibility is that putrescine and spermidine may act antagonistically to maintain a labile equilibrium between ribosomal subunits and vacant 70 S ribosomes. It may be significant that the putrescine effect is observed at the concentration of Mg2+ found to be optimum for initiation.     

Methylglyoxal bis(guanylhydrazone) elimination of polyamine effects on protein synthesis

The effect of methylglyoxal bis(guanylhydrazone) (MGBG), a structural analog of polyamines, on protein synthesis has been studied in the presence and absence of spermidine. The spermidine stimulation of polyphenylalanine- and MS2 RNA-directed RNA replicase synthesis in an Escherichia coli cell-free system and of globin synthesis in a rabbit reticulocyte cell-free system disappeared with the addition of MGBG. The spermidine reduction of misincorporation of leucine during polyphenylalanine synthesis in both E. coli and wheat germ cell-free systems was also disturbed by MGBG. MGBG noncompetitively interfered with polyamine stimulation of polyphenylalanine and globin synthesis, suggesting that MGBG could bind to both RNA and the complex of RNA and polyamine. MGBG was preferentially bound to ribosomal RNA among ribosomal RNA, poly(U), and calf thymus DNA, and strongly inhibited the amount of polyamine bound to ribosomal RNA. These results suggest that MGBG elimination of polyamine effects on protein synthesis may occur through the disturbance of polyamine binding to ribosomal RNA.     

The 60S ribosomal subunit is altered in the skeletal muscle of dystrophic hamsters

Polysomes from the skeletal muscle of normal and dystrophic hamsters were dissociated into ribosomal subunits by treatment with puromycin and the subunits from both strains were reassociated in all possible combinations. When their protein synthesis activity was assayed in a poly(U)-directed cell-free system at a low magnesium concentration, the reassociated ribosomes from dystrophic hamsters were less active than the ribosomes from control animals. The ribosomal defect is a property of the 60S subunit and is due to a ribosomal component rather than to abnormal binding of a non-ribosomal protein.     

Increase of degree of spermidine stimulation of polypeptide synthesis in the presence of phosphate

The addition of phosphate caused an increase in the degree of spermidine stimulation of polypeptide synthesis in an Escherichia coli and a wheat germ cell-free system. Optimal stimulation of polypeptide synthesis was observed at 20 mm phosphate for both systems, but concentrations of phosphate up to 40 mm had no additional effect. The increase of degree of spermidine stimulation in the presence of phosphate in an E. coli cell-free system occurred at the level of aminoacyl-tRNA binding to ribosomes and not at the level of peptide bond formation, translocation, or aminoacyl-tRNA formation. From the results of studies on RNase A sensitivity of ribosomal subunits and the effect of antibiotics known to act on the 30 S ribosomal subunits, it is suggested that the nature of the 30 S ribosomal subunits is changed by phosphate so that the degree of spermidine stimulation of polypeptide synthesis is increased.     

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.     

Mammalian mitochondrial ribosomes. Studies on the exchangeability of polypeptide chain elongation factors from bacterial and mitochondrial systems

Mammalian mitochondrial ribosomes from rat liver synthesised poly(phenylalanine) from [14C]-Phe-tRNA in the presence of a homologous 10(5) X gav supernatent fraction. The activity depended on the addition of synthetic template and was resistant to cycloheximide. The polyanion spermidine had a stimulatory effect on peptide synthesis in vitro. In contrast to Escherichia coli ribosomes, which also functioned with heterologous supernatant fractions, 55-S mitochondrial ribosomes were inactive when supplemented with heterologous supernatant fractions from E. coli or with purified bacterial elongation factors. EF-T slightly stimulated polyphenylalanine synthesis when added in combination with mitochondrial supernatant fractions. Two-dimensional electrophoretic analysis of the protein content of both supernatant fractions revealed considerable differences in the distribution of the species-specific proteins according to their isoelectric points. The mitochondrial supernatant proteins were in general more basic, and the few acidic proteins did not co-migrate with EF-Tu or EF-G from E. coli.     

Expression of different coding sequences in cell-free bacterial and eukaryotic systems indicates translational pausing on Escherichia coli ribosomes

Five different coding sequences of bacterial or eukaryotic origin in plasmids under the T7 promoter were expressed in a cell-free system derived from Escherichia coli. Translation on E. coli ribosomes resulted in a full-length product only in four of the five coding sequences tested. A unique pattern of less than full-length polypeptides was generated in each case. Many of these polypeptides on E. coli ribosomes reacted with a puromycin derivative, cytidylic acid-puromycin, which was radioactively labeled. Thus these incomplete polypeptides can be defined as nascent peptides bound to the ribosomal P site. Certain nascent peptides could be shifted into full-length protein indicating that they resulted from translational pausing. In contrast to these results, expression of the same coding sequences in a wheat germ or reticulocyte cell-free system resulted in a 80-90% full-length product with no evidence for nascent polypeptides and translational pausing.     

In vitro binding of 70S ribosomes to membrane structures of Escherichia coli

It was found that the maximal disattachment of the ribosomes from the membrane structures is observed upon their treatment with 10 mM tris-HCl buffer, pH 7.5, containing 250 mM sucrose, 750 mM KCl, 5 mM magnesium acetate and 1 mM EDTA or puromycin. The most effective attachment of ribosomes to the membrane occurs in 10 mM tris-HCl buffer, pH 7.5, containing 5% sucrose and Mg2+. The increase of Mg2+ concentration in the medium from 0.5 mM up to 1 mM results in a 2-fold increase of the ribosomes bound to the membranes. The concentration of the ribosomal material involved in the reaction is very essential for ribosome binding to the membranes. The amount of ribosomes bound to the membranes increases proportionally to the increase of the ribosome concentration in the reaction mixture.     

A high-yield cell-free system of protein synthesis of mouse liver

1. A fractionated cell-free system of protein synthesis has been developed from mouse liver. It is composed of polysomes, "pH 5" fraction, Mg2+, K+, ATP and a ATP generating system. 2. It operates optimally at 30-37 degrees C, in the presence of 4 mM MgCl2 and 90 mM KCl. 3. Spermine is highly inhibitory, while spermidine shows a bimodal action, in that submillimolar concentrations stimulate, while millimolar concentrations inhibit protein synthesis. 4. Both spermine and spermidine show an interesting selectivity, in that, even though they inhibit incorporation of amino acids into most proteins, they stimulate incorporation into a few proteins. 5. The system can be rendered mRNA-dependent, either by preincubation or by treatment with micrococcal nuclease. In both cases globin mRNA as well as TMV RNA are faithfully translated. 6. Compared to other published mammalian fractionated cell-free systems, the mouse liver system is more efficient by approximately one order of magnitude, since the rate of incorporation of leucine per min is 30 pmol/mg protein or 435 pmol/mg RNA or 1 mol/mol ribosomes.     

Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labelling of the ribosomal mRNA binding site.

Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.     

Globin mRNA translation on Artemia salina ribosomes with components from Friend leukemia cells.

Globin mRNA can be translated with relatively high efficiency in a fractionated cell-free system containing ribosomes prepared from cytst of Artemia salina. These ribosomes have unusually low endogenous activity for peptide synthesis in the absence of added mRNA. The system requires components from the postribosomal supernatant and from the 0.5 M KCl ribosomal wash fraction. Both these fractions were derived from either rabbit reticulocytes or unstimulated Friend leukemia cells that produce little or no hemoglobin. The activity of mRNA and enzyme fractions from rabbit reticulocytes and Friend leukemia cells were tested in this system in vitro for their ability to direct the synthesis of the alpha and beta chains of globin. The alpha:beta chain ratio synthesized from mRNA in the rabbit reticulocyte salt wash fraction was 4:1. The corresponding value for the 9-S mRNA fraction from the salt-washed reticulocyte ribosomes was 1:4, thus these two fractions appear to provide sources enriched in either alpha or beta globin mRNA. Under all conditions tested, the ratio and amounts of peptides formed in vitro appear to reflect mRNA composition. Globin mRNA from dimethysulfoxide-stimulated Friend leukemia cells when translated in vitro produced alpha and beta chains in a ratio of 1:1. These peptides are formed in the same ratio in the intact cells.     

GTP and Protein Synthesis in Cell-Free Systems

Abstract

Two supernatant fractions, T1 and T2, have been isolated and partially purified from rat liver and rabbit reticulocytes. In a cell-free system programmed with polyuridylic acid, both fractions are needed for phenylalanine polymerization. T1 factor and GTP are required for the enzymatic binding of phenylalanil-tRNA to template charged ribosomes. In the course of the binding reaction, GTP is hydrolyzed while no dipeptide formation can be detected. T2 factor coincides with a ribosome-linked GTPase, which is not stimulated by the addition of polyuridylic acid and phenylalanil-tRNA.     

Decomposition of ribosomal particles in Escherichia coli treated with mitomycin C

Exposure of cells of Escherichia coli to mitomycin C (5 mug/ml) resulted in a marked change in the sedimentation profiles of the cell-free extracts, indicating a specific decomposition of ribosomal particles. When the extracts were prepared in the presence of 0.01 m Mg(++) and analyzed by sucrose density gradient centrifugations, the 100S fraction disappeared rapidly from the treated cells. The 70S ribosomes were also degraded, but more slowly, with a concomitant accumulation of a fraction having a sedimentation coefficient of about 50S. However, decomposition of the 70S ribosomes was preceded by an almost complete loss of the 50S ribosomal subunits, as revealed by sedimentation analyses in the presence of 10(-4)m Mg(++). Synthesis of the ribosomes in the treated cells was also suppressed, being demonstrated by a lower incorporation of uracil-2-(14)C into the ribosomal fractions. However, the change in the ribosomal profile in the treated cells apparently resulted from the decomposition of pre-existing ribosomes, rather than from the inhibition of the net synthesis of ribosomes. Sedimentation analyses and chromatography of the nucleic acids extracted from the treated cells indicated extensive but delayed degradation of the ribosomal ribonucleic acid (RNA), but not of the soluble RNA or deoxyribonucleic acid fractions. Altered structure of the ribosomes in the treated cells was also indicated by their lower melting temperature, broadened thermal profile, higher electrophoretic mobility, and extreme sensitivity to ribonuclease treatment, compared with normal ribosomes. The synthesis of messenger RNA was inhibited progressively with time in the treated cells.