Presence Of Polyuridylic Acid Research Articles

Template-directed oligomerization of 5'-deoxy-5'-nucleosideacetic acid derivatives.

5'-Deoxy-5'-nucleosideacetic acids II-V are isostructural analogues of nucleotides with a carboxylate group in the place of the 5'-phosphate group. We have studied their oligomerization in aqueous solution using a water-soluble carbodiimide as the condensing agent in the presence or absence of an appropriate polynucleotide template. Condensation of adenylic acid analogues IIa, IIIa, and Va in the presence of polyuridylic acid were found to be the most efficient reactions. Cyclization of the activated monomers to lactones and the insolubility of the oligomers in aqueous solution were found to be obstacles to the efficient formation of long oligomers.

Role of tissue specific factors in the translation of brain messenger ribonucleic acids in vitro

The activity of brain ribosomal subunits was examined by measuring (a) the saturation of ribosomes with nascent polypeptides and the rates of release of completed chains; (b) interaction of the subunits with brain messenger RNA to form polysomal aggregates; (c) formation of polyphenylalanine in the presence of polyuridylic acid at high magnesium concentration and (d) inhibition by aurine tricarboxylic acid. The results showed that a portion of the subunits were defective in forming initiation complexes with brain messenger RNA, but translated polyuridylate efficiently. The subunits that did form polysomes were more competent than the heterologous systems (derived from Krebs ascites cells, reticulocytes or wheat germ) in carrying out reinitiations of brain mRNA translation. Both the homologous and the heterologous systems translated brain mRNA and synthesized the two brain specific proteins S-100 and the neuron specific enolase, indicating that each of the systems had all the necessary factors. However, homologous initiation factors, aminoacyl-tRNA synthetases and transfer RNAs were more effective, particularly at suboptimal concentrations. Our results suggest that discriminative translation of brain messenger RNA may take place based on relative proportions of required components in the reaction milieu rather than by the presence or absence of one or more special messenger RNA specific factors.

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

AbstractThe 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.

Experimental cardiac hypertrophy and the synthesis of poly (A) containing RNA and of myocardial proteins in the rat: the effect of digitoxin treatment.

The synthesis of poly(A) containing RNA was increased in heart of non-digitalized and digitalized rats after aortic constriction, the increase being of the same degree as that of the RNA lacking this sequence. No differences were found, either in the absence or presence of polyuridylic acid, in the incorporation of radioactivity into protein by cardiac ribosomes isolated from animals treated differently. It may be concluded, that after the constriction of the aorta the synthesis of mRNA proceeds at a similar rate as that of the bulk RNA, and that the treatment of the animals with digitoxin does not abolish the stimulus for hypertrophy.

Escherichia coli membrane proteins with an affinity for deoxyribonucleic acid

From the membrane fraction of Escherichia coli K-12 strain, four protein fractions (peaks I, IIa, IIb, and III) which have affinity for deoxyribonucleic acid (DNA) have been isolated. The molecular weights of these proteins are between 12,000 and 8,000. Only the peak III fraction contains a protein that binds preferentially to single-stranded DNA, whereas the others contain proteins that bind also to double-stranded DNA. The binding activity of the peak IIb protein is inhibited in the presence of polyuridylic acid. Peak I and peak IIa protein fractions behave like hydrophobic proteins.

Inhibited initial rates of poly-uridylic acid-directed phenylalanine incorporation by free ribosomes from the liver of rats fed hepatocarcinogens

Female Holtzman rats were fed diets containing 3′-methyl-4-dimethyl-aminoazobenzene (3′-Me-DAB) for 4 weeks; control rats were fed the basal diet. Free ribosomes and membrane-bound ribosomes were isolated from livers and initial rates of phenylalanine incorporation in vitro into proteins were determined both in the absence and presence of poly-uridylic acid (poly-U). Initial rates of protein synthesis by membrane-bound ribosomes were not detectably affected by 3′-Me-DAB ingestion. Although initial rates of free ribosomes from control rat livers were stimulated by poly-U additions, rates for free ribosomes from 3′-Me-DAB-fed rats were not appreciably changed by poly-U. Defective poly-U-directed phenylalanine incorporation by free ribosomes from rats fed 3′-Me-DAB: (a) was dependent upon time-on-diets; (b) was not an artifact associated with the method of isolating free ribosomes; (c) was not corrected by changing the magnesium level of reaction mixtures or by supplementing the levels of pH 5 precipitate fraction, elongation factors or initiation factors; and (d) was not due to a failure to bind poly-U. Feeding other hepatocarcinogens, viz. 4′-fluoro-DAB, 2-acetylaminofluorene, aflatoxin and thioacetamide, to female Holtsman rats of feeding any of these five hepatocarcinogens to male Fischer rats also led to the development of defective poly-U-directed phenylalanine incorporation by hepatic free ribosomes. Male Holtzman rats and female Fischer rats developed the defect more slowly, i.e. 8 weeks of feeding was necessary before the defect was detected with three of the five hepatocarcinogens. The possibility that defective poly-U-directed phenylalanine incorporation was due to a ribosomal alteration, and the possibility of a linkage between this phenomenon and the animal's risk for cancer were discussed.

Modification of E. coli ribosomes and coliphage MS2 RNA by bisulfite: effects on ribosomal binding and protein synthesis.

The reaction of E. coli 70s ribosomes with 0.2 M NaH-35 s03 (pH 7.1, 3.5hrs, 37 degree) led to the conversion of 4.5% of the uracil residues of the R, RNA into 5.6-dihydrouracil-6-sulfonate residues. The modified ribosomes exhibited a significant decrease in their ability to bind (14-C)-phenylalanyl-(RNA-phe and to incorporate (14-C)-phenylalanine into protein in the presence of polyuridylic acid. The ability of the modified ribosomes to form an initiation complex as measured by the A-U-G or coliphage MS2 RNA dependent binding of (14-C)-fmet-tRNA-fmet was also impaired, as was their ability to incorporate (14-C) lysine into protein with MS2 RNA as messenger. Treatment os MS RNA with 0.2 M sodium (35-S) bisulfite, pH 7.0 at 25 degrees C resulted in the substitution of 2.7% and 6.2% of the uracil residues by bisulfite after 1 and 3.5 hrs of reaction, respectively. Impairment of function of the MS2 RNA in both initiation complex formation and transplantation assays was observed. These reactions of uracil residues of mRNA and rRNA may be a cause of biological damage inflicted by sodium bisulfite and sulfur dioxide.

Condensation of amides of oligodesoxyadenylates in presence of polyuridylic acid

1. Various terminal amides of the di- and tridesoxynucleotide 5'-phosphates can be used for the chemical matrix synthesis of oligodesoxynucleotides. The principal reaction is dimerization. 2. The yields of the dimers decrease sharply if the amides of the dinucleotides are used as the starting materials.

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 MgCl(2). 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 [(14)C]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.

Binding of dihydrostreptomycin to Escherichia coli ribosomes: kinetics of the reaction.

Investigations were carried out on the binding of dihydrostreptomycin to purified (and reassociated) 70S ribosomes and 30S subunits from streptomycin-susceptible strains, and the results were compared with those of similar studies with native (run-off) 70S ribosomes. At 0 C, only a small fraction of purified 70S ribosomes and 30S sub-units bound 1 molecule of the antibiotic tightly, and at a rate comparable to the binding occurring with native 70S ribosomes. At temperatures of 10 C and above, there was a temperature-dependent increase in the extent of antibiotic binding to purified 70S and 30S particles up to a maximum of 1 molecule/ribosomal particle, but the kinetics of binding was slow in comparison to that taking place at 0 C. These and other results suggest that a major fraction of 30S subunits and purified (or reassociated) 70S ribosomes are inactive in binding the antibiotic. This has been localized to an instability of the free 30S subunit, which in solution at 0 C has a half-life of 5 hr or less. Inactive 30S or 70S particles could be thermally activated, with the latter being identical in their streptomycin-binding properties to native 70S ribosomes. The activation kinetics were slow in comparison to the binding kinetics for the antibiotic and were indicative of a conformational change in ribosomal structure. There thus appears to be a reversible transition between active and inactive forms of the ribosomal particles for streptomycin binding, but additional binding sites for the antibiotic are not created by the transitions. The active form of the 30S subunit can be stabilized in the presence of polyuridylic acid, but much more effectively by association with the 50S subunit to form a 70S ribosome. The kinetics of dihydrostreptomycin binding were studied in both directions of the reaction, and the reaction in the direction of binding was found to be several orders of magnitude faster than that of the reverse, or debinding, direction. The kinetics of the exchange of bound dihydrostreptomycin with the free antibiotic were also determined and shown to have rate constants that are very similar to those of the debinding reaction, which is the rate-limiting step. It appears likely that the exchange reaction is proceeding via the same reaction pathway. The temperature dependence of the kinetics of dissociation of the bound complex was much greater than that in the direction of binding and accounted for most of the temperature dependence of the binding equilibrium. From the determined thermodynamic and activation parameters, it appears likely that binding of the antibiotic induces a conformational change in ribosomal structure to one that is less ordered than the native particle. Heterogeneity has been found in the kinetics of binding and of exchange, with a fraction of the 70S population showing slower kinetics for both directions of the reaction.

Peptidyl transferase: A new method for kinetic studies

The reaction of N-Acetyl-phenylalanyl-t-RNA with E. coli ribosomes in the presence of poly Uridylic acid, guanosine 5′-triphosphate, and initiation factors (ribosomal wash), leads to the formation of N-Acetyl-phenylalanyl-RNA-ribosome-poly Uridylic acid complex, which can be isolated on a cellulose nitrate filter. The isolated complex retains its ability to react with puromycin producing N-Acetyl-phenylalanyl-puromycin. The formation of N-Acetyl-phenylalanyl-puromycin follows simple enzyme kinetics with respect to puromycin giving linear Lineweaver-Burk plots. A number of inhibitors of peptidyl transferase inhibit this reaction. The reaction can also be used to study the nature of the complex formed and the factors or conditions which modify its formation.

Template catalysis of acetyl transfer reactions

Acetyl adenylate, 2′(3′)-O- acetyl adenosine and 5′-O- acetyl adenosine, like adenosine itself, form ordered helices with polyuridylic acid in the presence of Na + and Mg 2+. The transfer of acetate from acetyl adenylate to [8- 14C]adenosine is facilitated in the presence of polyuridylic acid and occurs specifically to the 2′(3′)-position. Under the same condition, 2′(3′)-O- acetyl adenosine transfers acetate to the 5′-position of adenosine. However, 5′-O- acstyl adenosine transfers acetate to both the 2′(3′)- and 5′-positions of adenosine. These results establish that a polyuridylic acid template is capable of orienting adenosine derivatives in such a way as to bring about highly specific intramolecular acetyl transfer reactions.

The reverible dissociation and activity ofribosoma subunits of liver and skeletal muscle from rats.

Ribosomal subunits were prepared from rat liver and skeletal muscle after incubation with puromycin and treatment at low concentrations of Mg(2+). After isolation the resulting subunits could be recombined and the particles effected the synthesis of polyphenylalanine in the presence of polyuridylic acid. Hybrid particles formed from subunits of liver and muscle respectively were also active. The homogeneity of the isolated subunits was checked by polyacrylamide-gel-agarose electrophoresis. The method is shown to be a reliable and comparatively simple way of preparing active ribosomal subunits from skeletal muscle.

Transfer of amino acids from aminoacyl-tRNA into protein by isolated ribosomes of the house cricket

The optimal conditions for the transfer of amino acids from aminoacyl-tRNA into protein by Acheta domesticus ribosomes have been defined. In general, the properties and requirements are analogous to those of systems prepared from other eukaryotes. The activity of the ribosomes is enhanced when they are supplemented with polymerizing enzymes isolated from chick embryos or from rat liver. The system is completely inhibited by ribonuclease; puromycin and cycloheximide are also strongly inhibitory. Endogenous incorporation is not eliminated by pre-incubating the ribosomes under optimal conditions for protein synthesis. However, pre-incubation in the presence of puromycin yields ribosomes devoid of endogenous incorporating activity. Such ribosomes are capable of phenylalanine incorporation in the presence of polyuridylic acid, and may be dissociated into sub-units, without the use of high KCl concentrations, in the presence of 1 mM Mg 2+.

Peptidyl transferase activity of [formula omitted] ribosomes having an altered protein component in the 50S subunit

Ribosomes from the mutants of Escherichia coli having an altered specific 50S ribosomal protein component showed much the same ability to bind N-acetylphenylalanyl-tRNA in the presence of polyuridylic acid but were found to have distinctly lower peptidyl transferase activity than those from parent Q13 cells. The ribosomes “less active” in peptidyl transferase activity have been shown to be convertible to the “active” state by changing the environmental K + or NH 4 + concentration.

Purification and Partial Characterization of the Aminoacyl Transfer Ribonucleic Acid Binding Enzyme from Rabbit Reticulocytes

Abstract The aminoacyl transfer RNA binding enzyme has been purified to apparent homogeneity from rabbit reticulocytes. The active protein is a single molecular species. It catalyzes the guanosine triphosphate-dependent binding of phenylalanyl transfer RNA to ribosomes in the presence of polyuridylic acid, has activity as a ribosome and aminoacyl transfer RNA-dependent guanosine triphosphatase, and is required for the synthesis of peptides from aminoacyl transfer RNA. The protein has a molecular weight of 186,000 and an s20,w of 6.7 S. In the presence of dissociating agents (8 m urea or 5 m guanidine-HCl) the protein undergoes dissociation into subunits of molecular weight 62,000. The results indicate that the native protein consists of three, possibly identical, subunits.

Active hybrid 80 s particles formed from subunits of rat, rabbit and protozoan ( Tetrahymena pyriformis) ribosomes

Ribosomes of rat skeletal muscle and rat liver were dissociated in 880 m m-potassium chloride at 28 °C in the presence of magnesium ions (12.5 m m); at 4 °C the subunits formed aggregates. In contrast, the subunita formed from rabbit muscle and Tetrahymena pyriformis ribosomes in the same ionic circumstances did not aggregate at 4 °C. The ability of rat muscle ribosomal subunits to combine with subunits of other 80 s ribosomes was determined by density-gradient analysis and by estimation of polyuridylic acid-directed polypeptide synthesis. Both hybrids formed and were active when rat muscle and rat liver subunits were combined. Subunits from rat and rabbit skeletal muscle ribosomes also formed active hybrids, despite a difference in the 60 s particles of the two species; the rat 60 s subunit has a far greater tendency to form 90 s dimers at low temperatures. However, only one hybrid was detected by density-gradient analysis when rat muscle and Tetrahymena subunits were mixed. The small subunits of the protozoan and the large subunits of rat formed 80 s particles, which synthesized polyphenylalanine at the same rate as re-associated Tetrahymena ribosomes. The 40 s of rat and the 60 s of the protozoan did not appear to form 80 s particles, but the mixture had some protein synthetic activity in the presence of polyuridylic acid. The ribosomal proteins contained in the subunits of rat muscle and Tetrahymena ribosomes were markedly different.

Reversible Effect of Lithium Chloride on Ribosomes

Abstract The binding of aminoacyl transfer RNA to ribosomes either in the presence (specific binding) or in the absence (nonspecific binding) of messenger RNA was inhibited by LiCl. This effect was partially reversible by NH4+ or K+. The ribosomes were completely dissociated into 50 S and 30 S subunits in the presence of 0.2 m LiCl. When LiCl was removed, the subunits associated again. The activity of ribosomes to synthesize polyphenylalanine in the presence of polyuridylic acid was recovered upon removal of LiCl. This reversible effect of LiCl was antagonized by the presence of a low concentration of NH4+.

Effect of varying the KCl and MgCl 2 concentration on the enzymic and nonenzymic binding of phenylalanyl-RNA to reticulocyte ribosomes

Binding of phenylalanyl- 3H-RNA to reticulocyte ribosomes in the presence of polyuridylic acid was studied using various concentrations of MgCl 2 and KCl. At salt conditions optimal for synthesis of polyphenylalanine (6.7 m m MgCl 2 and 67 m m KCl) a soluble fraction (binding enzyme) and guanosine 5′-triphosphate (GTP) were required for binding to occur. At higher concentrations of MgCl 2 or lower concentrations of KCl binding could occur without GTP and enzyme. Some of the phenylalanyl-RNA bound enzymically could be converted to diphenylalanine by incubation of the isolated ribosomes with a second enzyme (peptide synthetase). Phenylalanyl-RNA bound nonenzymically could be converted to the dipeptide only when GTP and the binding enzyme were included in this second incubation. A model of the ribosome with two binding sites for sRNA, the acceptor and donor sites, can be used to explain these results. Nonenzymic binding may occur predominantly, though not exclusively, in the acceptor site. Increasing the molar ratio of KCl to MgCl 2 could, as the result of changes in the ribosome shape, allow binding to occur readily in both sites; now GTP and the binding enzyme are required, perhaps to aid in the translocation of phenylalanyl-RNA from the acceptor to the donor site. A peptide bond may be formed between two phenylalanyl-RNAs in adjacent sites on the same ribosome. In the events which are prerequisite to formation of this first peptide bond, GTP and the binding enzyme appear to have another role in addition to aiding the physical binding of phenylalanyl-RNA to the ribosomes.

Adsorption chromatography of phenylalanine peptides on Sephadex

The adsorption properties of dextran gels have been utilized to separate the first six members of an homologous series of phenylalanine peptides, the longer members being more highly retarded. Separation is influenced by gel structure, pH and the ionic environment. This technique has been used to verify that such homologous peptides are synthesized by Escherichia coli extracts in the presence of polyuridylic acid.