A decreased aminoacyl-transfer-ribonucleic acid-binding capacity of 40S ribosomal subunits resulting from hypophysectomy of the rat

A technique that permitted the reversible dissociation of rat liver ribosomes was used to study the difference in protein-synthetic activity between liver ribosomes of normal and hypophysectomized rats. Ribosomal subunits of sedimentation coefficients 38S and 58S were produced from ferritin-free ribosomes by treatment with 0.8m-KCl at 30 degrees C. These recombined to give 76S monomers, which were as active as untreated ribosomes in incorporating phenylalanine in the presence of poly(U). Subunits from normal and hypophysectomized rats were recombined in all possible combinations and the ability of the hybrid ribosomes to catalyse polyphenylalanine synthesis was measured. The results show that the defect in ribosomes of hypophysectomized rats lies only in the small ribosomal subunit. The 40S but not the 60S subunit of rat liver ribosomes bound poly(U). The only requirement for the reaction was Mg(2+), the optimum concentration of which was 5mm. No apparent difference was seen between the poly(U)-binding abilities of 40S ribosomal subunits from normal or hypophysectomized rats. Phenylalanyl-tRNA was bound by 40S ribosomal subunits in the presence of poly(U) by either enzymic or non-enzymic reactions. Non-enzymic binding required a Mg(2+) concentration in excess of 5mm and increased linearly with increasing Mg(2+) concentrations up to 20mm. At a Mg(2+) concentration of 5mm, GTP and either a 40-70%-saturated-(NH(4))(2)SO(4) fraction of pH5.2 supernatant or partially purified aminotransferase I was necessary for binding of aminoacyl-tRNA. Hypophysectomy of rats resulted in a decreased binding of aminoacyl-tRNA by 40S ribosomal subunits.     

Effect of polaymines on yeast cell-free protein synthesizing system. I. Influence of spermine and spermidine on aminoacyl-tRNA transfer reaction.

Spermine and spermidine added to a Saccharomyces cerevisiae cell-free protein synthesizing system increased phenylalanine polymerization reaction several-fold at suboptimal concentration of Mg2+ and approximately two-fold at optimal amounts of Mg2+. The addition of polyamines greatly stimulated the enzymatic and nonenzymatic binding of phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA to ribosomes. The binding of the acetylated derivative was higher than phenylalanyl-tRNA, however, as it was shown the former was bound exclusively to the A site of the ribosome. Contrary to the binding process, the puromycin reaction was not stimulated by spermine added at a concentration which enhanced the polyphenylalanine synthesis. These results indicate that polyamines have not only a sparing effect on the Mg2+ requirement for yeast protein synthesis in vitro and suggest that one of the possible sites of polyamines action might be the binding of aminoacyl-tRNA to ribosomes.     

IN VlTRO BINDING OF PHENYLALANYL-tRNA TO NEONATAL AND ADULT MOUSE BRAIN RIBOSOMES

The ability of brain ribosomes, isolated from mice of various ages, to bind phenylalanyl-tRNA was measured under various reaction conditions. In the presence of template RNA (polyuridylic acid) the binding could be measured by both enzymic and non-enzymic assays. In general, the binding requirements for the brain system were similar to those previously described for microbial and eukaryotic systems. Although previous studies have shown that ribosomes obtained from increasingly older mow brain tissue were less active in polyphenylalanine synthesis, no significant differences in phenylalanyl-tRNA binding to polysome complexes could be detected. The binding of phenylalanyl-tRNA by ribosomes isolated from both neonatal and mature mouse brain tissue was similar with regard to GTP and polyuridylic acid dependence, magnesium ion concentration and reaction kinetics. Similar binding of phenylalanyl-tRNA by young and mature brain ribosomes was also measured with ribonucleoprotein particles previously stripped with puromycin. The results are discussed in light of the rapid alteration of macromolecular synthesis during postnatal brain development and the possible role of the interaction between ribosomes and tRNA.     

The attachment of polyuridylic acid to reticulocyte ribosomes

The attachment of polyuridylic acid to reticulocyte ribosomes was studied by using polyadenylic acid, which inhibits the attachment reaction only, while permitting translation of polyuridylic acid bound to ribosomes. After addition of polyadenylic acid the amount of polyphenylalanine synthesized under standard conditions was taken as a measure of the bound polyuridylic acid. In this way certain parameters of the attachment reaction and the subsequent translation of attached polyuridylic acid were defined: (1) polyuridylic acid-ribosome interaction at 37 degrees requires only Mg(2+) at an optimum concentration of 8mm; (2) K(+) (required for translation) is a non-competitive inhibitor of the attachment reaction; (3) optimum polyphenylalanine synthesis directed by attached polyuridylic acid occurs at 5mm-Mg(2+) concentration; (4) from kinetic studies single ribosomes appear to participate in the attachment reaction.     

Evidence for specific inhibition of translocation of aminoacyl-transfer ribonucleic acid by the pretreatment of ribosomes of Bacillus subtilis with p-chloromercuribenzoate

The biological activities of Bacillus subtilis ribosomes pretreated with 0.5, 1, or 2 mmp-chloromercuribenzoate (PCMB) were examined. The ribosomes treated with 1 or 2 mm PCMB lost their capacity to synthesize polyphenylalanine as well as the capacity to bind phenylalanyl-transfer ribonucleic acid. On the other hand, approximately 70% of the polyphenylalanine synthesis capacity was lost with ribosomes pretreated with 0.5 mm PCMB. This inhibition was found to be due to a specific inhibition of the translocation step. The effect of 0.5 mm PCMB was reversed by 20 mm Mg(2+) without the loss of PCMB bound to ribosomes, while beta-mercaptoethanol partially reversed the inhibitory effect with concomitant loss of bound PCMB.     

The ribosomal binding site for peptidyl-transfer-ribonucleic acid

The properties of a site, on Escherichia coli ribosomes, which binds peptidyl-s-RNA (where s-RNA refers to ;soluble' or transfer RNA) have been investigated. The binding is stable both in low Mg(2+) concentration (0.1mm), and in high Mg(2+) concentration (10mm) in the absence or presence of potassium chloride (86mm). Puromycin has been used to break the bond between the s-RNA and the polypeptide, and in the absence of further protein synthesis this technique exposes free s-RNA molecules on the ribosomes. The s-RNA exposed remains bound in high Mg(2+) concentration, but the binding is unstable in high Mg(2+) concentration with potassium chloride and the s-RNA can be freed completely from the ribosomes by lowering the Mg(2+) concentration. It can also be displaced by s-RNA in the medium. It is suggested that this ribosomal binding site for peptidyl-s-RNA is the site for peptide bond formation. Further, it is proposed that it is the same site that can be demonstrated on ribosomes not engaged in protein synthesis and that, in high Mg(2+) concentration, will bind s-RNA molecules charged or uncharged with amino acids.     

Binding and translation of turnip-yellow-mosaic-virus ribonucleic acid by skeletal-muscle ribosomes from normal and diabetic rats

Ribosomes from skeletal muscle of diabetic rats were less active than normal ribosomes in protein synthesis directed by turnip-yellow-mosaic-virus RNA. The proportion of ribosomes from muscle of diabetic rats capable of binding turnip-yellow-mosaic-virus RNA was greater than normal, but there was no difference in the equilibrium constants for the binding reaction. The turnip-yellow-mosaic-virus RNA was bound preferentially to the small (40S) ribosomal subunit, whereas the decrease due to diabetes in its translation was associated with the large (60S) subunit. Thus the diminished capacity of ribosomes from muscle of diabetic rats to translate turnip-yellow-mosaic-virus RNA was not the result of decreased binding of the template.     

Protein synthesis in the cotyledons of Pisum sativum L. Protein factors involved in the binding of phenylalanyl-transfer ribonucleic acid to ribosomes

1. Proteinaceous factors contained in a 0.5m-KCl extract of ribosomes from pea cotyledons form a ternary complex at 0 degrees C with [(14)C]phenylalanyl-tRNA and poly(U). The complex is measured by its quantitative retention on Millipore filters. 2. Complex-assembly is optimal at 5mm-Mg(2+) and is independent of GTP and ribosomes. 3. The addition of ribosomes is required to stabilize the complex at 34 degrees C. The complex binds to a puromycin-sensitive site on the ribosome. 4. Soluble factors from the 250000g supernatant of pea cotyledon form a Millipore-retainable complex dependent on GTP and ribosomes. 5. Complex-formation by soluble factors has a Mg(2+) optimum of 10-12mm and forms a puromycin-insensitive complex with ribosomes. 6. The function of the ribosomal protein factors and the supernatant fraction in initiation of protein synthesis is discussed.     

The effect of hypermethylation on the functional properties of transfer ribonucleic acid. Ribosome-binding and polypeptide synthesis

1. Phenylalanyl-tRNA formed after chemical hypermethylation of Escherichia coli B tRNA was able to bind to ribosomes with the same efficiency as normal phenylalanyl-tRNA. 2. Under incubation conditions used in the ribosome-binding assay, hypermethylation of tRNA did not measurably decrease the stability of either inter-nucleotide phosphodiester bonds or the covalent bond between amino acid and tRNA in phenylalanyl-tRNA. 3. The ability of hypermethylated tRNA to take part in polyphenylalanine synthesis was inhibited progressively as the degree of hypermethylation increased. 4. Hypermethylation of tRNA affected polyphenylalanine synthesis at the stage of amino acid recognition and at a further point in the synthesis but not at the level of codon-anticodon recognition. 5. The formation of polylysine was more seriously affected by hypermethylation of tRNA than would be accounted for by inhibition of amino acid acceptance alone. 6. Polyproline formation was completely inhibited by the presence of 7mol% excess of methyl groups in tRNA. 7. The possibility of a link between amino acid acceptance and ribosome-binding was suggested for phenylalanyl-tRNA, but not for lysyl- or prolyl-tRNA.     

Function of ribosomes and glutamyl-tRNA isoacceptors in protein synthesis in regenerating skeletal muscle

Ribosomes from 8-day-regenerating rat skeletal muscle have been shown to be more active in poly(U)-directed polyphenylalanine synthesis than ribosomes from control muscle. This difference persists after salt washing of the ribosomes and does not appear to be due to the presence of ribonuclease associated with the control ribosome population. Ribosomes from control muscle were also less active than those from regenerates in the nonenzymatic binding of phenylalanyl-tRNA to ribosomes and in the peptidyltransferase reaction. Three glutamyl-tRNA isoacceptors have been isolated from 8-day-regenerating rat skeletal muscle by preparative RPC-5 chromatography of total tRNA charged with [3H]glutamic acid. The two major isoacceptors observed, tRNAgluI and tRNAgluIII, respond to the glutamic acid codons GAG and GAA, respectively. A third, minor glutamyl isoacceptor, tRNAgluII, also responds to the codon GAA. When the three isoacceptors were tested for function in a polysomal cell-free protein synthesizing system, it was found that their relative levels of utilization were essentially identical to their relative abundances. Thus, the tRNA which increases in relative amount after the induction of regeneration, tRNAgluII, is not preferentially utilized for overall muscle protein synthesis.     

Monoclonal antibodies against acidic phosphoproteins P0, P1, and P2 of eukaryotic ribosomes as functional probes.

Mice were immunized against ribosomal acidic proteins P1 and P2 from Artemia salina, and three kinds of monoclonal antibodies were isolated. One recognized P0 in addition to both P1 and P2 (anti-P). The other two recognized either P1 (anti-P1) or P2 (anti-P2) specifically and did not recognize P0. The anti-P antibody, but not anti-P1 or anti-P2, recognized a 22-amino acid peptide corresponding to the carboxyl-terminal sequence common to P1 and P2. This antibody, but not the others, inhibited poly(U)-directed polyphenylalanine synthesis. The anti-P1 bound to ribosomes but failed to inhibit polyphenylalanine synthesis: the anti-P2 did not bind to ribosomes at all. The anti-P and its Fab fragments inhibited the elongation step of protein synthesis, namely, the binding of elongation factors 1 alpha and 2 to ribosomes as well as their ribosome-coupled GTPase activities. Anti-P had little effect on the nonenzymatic phenylalanyl-tRNA binding to ribosomes and on peptidyltransferase activity. These results suggest the functional importance of the homologous carboxyl-terminal region of the three P proteins for the interaction of the ribosome with the two elongation factors. The epitope of anti-P1 must reside in a region of the protein which is not directly involved in its function.     

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.     

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.     

Mechanism of stimulation of polyphenylalanine synthesis by spermidine.

It is shown that the stimulation of polyphenylalanine synthesis by spermidine is due mainly to the stimulation of initiation of polypeptide synthesis by following reasons: 1) the binding of poly(U) to ribosomes was stimulated more by spermidine than the binding of Phe-tRNA to ribosomes, and 2) the number of polyphenylalanine chains was increased more by spermidine than the extension of the chain length. In addition, it is shown that 30S ribosomal subunits are responsible for the stimulation of polyphenylalanine synthesis by spermidine.     

In Vitro Protein Synthesis and Aging in Rhizoctonia solani

A study was made of the ability of cell-free protein synthesis systems from vegetative cells of different age of the fungus Rhizoctonia solani to produce polyphenylalanine. Polyuridylic acid-directed phenylalanine incorporation into peptides decreased linearly with cell age. The 105,000 x g supernatant fluid and ribosomal fractions were equally responsible for the total loss of synthetic activity of the older cells. Initial rates of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase activity decreased with increasing cell age, which accounted for the defect of the supernatant fraction. An accelerated degradation of soluble phenylalanyl-RNA was associated with the ribosomes of the older cells. In vitro systems from cells of different age transferred phenylalanine from phenylalanyl-tRNA to polyphenylalanine at similar rates. Of the 15 specific aminoacyl-tRNA synthetases assayed, 5 increased and 5 decreased in specific activity with increased age; 3 others did not change during aging and 2 were below acceptable detectable levels.     

Cell-free protein synthesis by kidney from the aging female Fischer F344 RAT

This is the first report to describe and characterize a cell-free protein synthesis system derived from kidney tissue. The optimum conditions for [³H]valine incorporation into protein by the post-mitochondrial supernatant from whole kidneys were found to be: pH 6.9, 7.5 mM MgCl₂, 150 mM KCl, 10 mM ATP, and 2 mM GTP. The cell-free protein-synthetising activities of kidneys isolated from 4.5-, 7.5-, 22-, and 31-month-old female Fischer F344 rats were measured using the post-mitochondrial supernatant. A 73–87% decrease in cell-free protein synthesis was observed between 4.5 and 31 months of age. Both the cell sap and microsomal fractions of the kidney post-mitochondrial super-natant from old rats were less active in protein synthesis than these fractions from the kidneys of young rats. No age-related change in the activity of RNA-ase in the kidney post-mitochondrial supernatant was observed. Kidney ribosomes stripped of endogenous mRNA were found to be active in poly(uridylic acid)-directed polyphenylalanine synthesis. The effect of aging on the fidelity of translation was determined by measuring poly(uridylic acid)-directed [¹⁴C]-phenylalanine and [³H]leucine incorporation by kidney ribosomes isolated from rats of various ages. No age-related change in the fidelity of poly(uridylic acid) translation by kidney ribosomes was observed.     

Binding of periodate-oxidized guanine nucleotides to eukaryotic elongation factor 2

Periodate-oxidized guanine nucleotides (GTPox and GDPox) were shown to bind stoichiometrically to rat liver elongation factor 2 (EF-2). This binding was quantitatively inhibited in the presence of GTP. After binding, oxidized nucleotides remained on EF-2 despite extensive dialysis. They exchanged, however, with free quanine nucleotides in the course of prolonged (greater than 1 h) incubations. The prior reduction EF-2.GTPox with NaBH4 abolished, to a large extent, this slow exchange. Thus, a Schiff's base was implicated to be formed between EF-2 and oxidized guanine nucleotides. Mg2+ increased the GTPox concentration necessary for a stoichiometric binding to EF-2. EF-2-oxidized nucleotide conjugates bound in the presence of ribosomes a second molecule of GTP (or GTPox). GTPox bound to EF-2 in the presence of ribosomes appeared to exchange readily with free GTP. Moreover, GTPox proved to be active as substrate in EF-2 and ribosome-dependent GTPase reaction: Km values found for GTPox and GTP were 7.7 and 3.4 microM, respectively. The binding of GTPox to EF-2 inhibited only partially the subsequent ribosome-dependent GTP binding, and GTPase reaction or polyphenylalanine (polyPhe) synthesis. On the other hand, the binding of GuoPP[CH2]Pox to EF-2 inhibited all of these reactions strongly. The nature of the binding site involved in the direct interactions of EF-2 with guanine nucleotides is discussed in the light of these results.     

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.     

The binding of spermine to the ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus

1. The total intracellular concentrations of Na(+), K(+), Mg(2+), spermine, spermidine and RNA were measured in Bacillus stearothermophilus. 2. The binding of spermine to ribosomes and to ribosomal RNA from B. stearothermophilus was studied under various conditions by using a gel-filtration technique. 3. The affinity of spermine for ribosomes and for ribosomal RNA decreased with increasing ionic strength of the medium in which they were suspended. 4. The extent of spermine binding did not change appreciably in the temperature range 4-60 degrees . 5. Optimum binding occurred at about pH7.0. 6. The number of binding sites for spermine on either ribosomes or ribosomal RNA was 0.10-0.13/RNA phosphate group. 7. A high proportion of the intracellular spermine is likely to be bound to the ribosomes in vivo; spermine competes with Mg(2+) on equal terms for sites on the ribosomes.     

Purification and characterization of three elongation factors, EF-1 alpha, EF-1 beta gamma, and EF-2, from wheat germ

Three elongation factors, EF-1 alpha, EF-1 beta gamma and EF-2, have been isolated from wheat germ. EF-1 alpha and EF-2 are single polypeptides with molecular weights of approximately 52,000 and 102,000, respectively. The most highly purified preparations of EF-1 beta gamma contain four polypeptides with molecular weights of approximately 48,000, 46,000 and 36,000, 34,000. EF-1 alpha supports poly(U)-directed binding of Phe-tRNA to wheat germ ribosomes and catalyzes the hydrolysis of GTP in the presence of ribosomes, poly(U), and Phe-tRNA. EF-2 catalyzes the hydrolysis of GTP in the presence of ribosomes alone and is ADP-ribosylated by diphtheria toxin to the extent of 0.95 mol of ADP-ribose/mol of EF-2. EF-1 beta gamma decreases the amount of EF-1 alpha required for polyphenylalanine synthesis about 20-fold. EF-1 beta gamma enhances the ability to EF-1 alpha to support the binding of Phe-tRNA to the ribosomes and enhances the GTPase activity of EF-1 alpha. Wheat germ EF-1 alpha, EF-1 beta gamma, and EF-2 support polyphenylalanine synthesis on rabbit reticulocyte ribosomes as well as on yeast ribosomes.