Inhibition by ricin of protein synthesis in vitro: 60S ribosomal subunit as the target of the toxin (Short Communication)

Poly(U)-directed polyphenylalanine synthesis by rat liver ribosomes is strongly inhibited by ricin. Experiments involving hybridization between subunits derived from normal and ricin-treated ribosomes demonstrate that the 60S subunit is the site of action of the toxin. The toxin inactivates the 60S subunit independently of the presence of the 40S subunit.     

The cytotoxic activity of Shigella toxin. Evidence for catalytic inactivation of the 60 S ribosomal subunit

The cytotoxic test system for Shigella shigae toxin was improved and used to study the stability of the toxin to various pH values, temperature, and chemicals. Inhibition of protein synthesis is the first demonstrable effect in cells treated with Shigella toxin. This inhibition appears to be at the level of peptide chain elongation. An inhibition effect on cell-free protein synthesis is exhibited by toxin pretreated first with trypsin and then with dithiothreitol and 8 M urea or 1% sodium dodecyl sulfate. Ribosomes treated with toxin or its A1 fragment had lost most of their ability to polymerize [14C]phenylalanine in a poly(U)-dependent cell-free system. Salt-washed ribosomes in simple buffered solutions were inactivated at a rate of at least 40 ribosomes/(min) (A1 fragment). Addition of antitoxin immediately stopped further inactivation, but it did not reactivate the inactivated ribosomes. 60 S ribosomal subunits from toxin-treated ribosomes had a marked reduction in ability to support polyphenylanine synthesis, whereas 40 S subunits from toxin-treated ribosomes retained their activity. Toxin-treated ribosomes retained their ability to incorporate [3H]puromycin into growing peptide chains, indicating that the peptide bond formation is not the function inhibited.     

Functional heterogeneity of the 30 S ribosomal subunit of Escherichia coli. 3. Requirement of protein S1 for translation

Poly(U)-dependent polyphenylalanine synthesis is completely dependent on the presence of ribosomal protein S1. Polysomes generated under the direction of poly(U) contain approximately one molecule of S1 per ribosome. Isolation of 30 S ribosomes from poly(U)-generated polysomes by a procedure requiring a low concentration of Mg²⁺ (0·25 mM) results in loss of S1. S1 is probably also required for the phage RNA-dependent binding of formylmethionyl-tRNA. The data are discussed in relation to current concepts of the functional aspects of ribosome heterogeneity.     

On the mechanism of protein-synthesis inhibition by abrin and ricin. Inhibition of the GTP-hydrolysis site on the 60-S ribosomal subunit.

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell-free systems from rabbit reticulocytes and Krebs II ascites cells. In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl-tRNA, demonstrating that the toxins act at some point after the charging of tRNA. Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti-toxins were added. Recombination experiments between toxin-treated ribosomes and untreated supernatant factors and vice versa showed that the toxin-treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis. Recombination experiments between toxin-treated isolated 40-S subunits and untreated 60-S subunits and vice versa showed that only when the 60-S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited. Both the EF-1-catalyzed and the EF-2-catalyzed ability of the ribosomes to hydrolyze [gamma-32P]GTP was inhibited by abrin and ricin. An 8-S complex released from the 60-S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8-S complex were inhibited by abrin and ricin. The present data indicate that there is a common site on the 60-S subunits for EF-1- and EF-2- stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.     

Biochemical changes in hereditary progressive muscular dystrophies. Defect of protein synthesis in fibroblasts, muscle tissues and blood cells

80S ribosomes and ribosomal subunits were isolated from fibroblasts, muscle tissues and blood cells of patients with different muscular dystrophies (MD) as well as of controls and were used for in vitro measurement of ribosomal protein synthesis (RPS) in a poly(U)-directed polyphenylalanine synthesis system. The activity of ribosomes from the patients showed a disease-dependent decrease compared to normal controls. Examination of hybrid 80S ribosomes consisting of 40S and 60S subunits of patients and the corresponding control cells revealed that the loss of RPS activity was related to one or both of the ribosomal subunits depending on the type of MD.     

Isolation of ribosomal subparticles from human placenta containing intact rRNA and determination of the functional activity of the 80S ribosome

The method for isolation of human placenta ribosomal subunits containing intact rRNA has been determined. The method uses fresh unfrozen placenta. Activity of 80S ribosomes obtained via reassociation of 40S and 60S subunits in non-enzymatic poly(U)-mediated Phe-tRNAPhe binding, was near 75% (maximal [14C]Phe-tRNA(Phe) binding was 1.5 mol Phe-tRNA(Phe) per mol of 80S ribosomes). Activity of 80S ribosomes with damaged rRNA isolated from frozen placenta was 2 times lower (the maximum level of poly(U)-dependent Phe-tRNA(Phe) binding was 0.7 mol per mol of ribosomes). The activity 80S ribosomes in poly(U)-mediated synthesis of polyphenylalanine was determined by using fractionated ("ribosomeless") protein synthesising system from rabbit reticulocytes. In this system up to the 50 mol of Phe residues per mol of 80S ribosomes are incorporated in acid insoluble fraction in 1 hour, at 37 degrees C. The obtained level of [14C]phenylalanine incorporation is three times as much as the amount of Phe residues observed for the ribosomal subunits, isolated from frozen placenta.     

Inhibition of protein synthesis by ricin: experiments with rat liver mitochondria and nuclei and with ribosomes from Escherichia coli (Short Communication)

1. Ricin, a toxic protein from the seeds of Ricinus communis which inhibits poly(U)-directed polyphenylalanine synthesis by rat liver ribosomes (Montanaro et al., 1973), does not affect protein synthesis by isolated rat liver mitochondria. 2. The toxin is ineffective also on poly(U)-directed polyphenylalanine synthesis in reconstituted systems with ribosomes isolated from rat liver mitochondria or from Escherichia coli. 3. Ricin inhibits protein synthesis by isolated rat liver nuclei, but at concentrations much higher than those affecting rat liver ribosomes.     

Modification of 50S ribosomal subunits withN-bromosuccinimide

The 50S subunits ofEscherichia coli ribosomes were modified with the tryptophan reagentN-bromosuccinimide, and the sulfhydryl groups, the modification of which is accompanied by stimulation of polypeptide synthesis (López-Rivas, A. et al. (1978) Eur. J. Biochem. 92, 121), were regenerated by incubation with simple thiols. This treatment inactivates poly(U)-dependent polyphenylalanine synthesis, peptidyl transferase and elongation factor G-dependent GTPase. Incubation with proteins from untreated 70S ribosomes produces partial reactivation of polyphenylalanine synthesis and GTPase activity. Modification is accompanied by loss of 4–5 tryptophan residues per subunit.Abbreviation SucNBr N-bromosuccinimide     

Isolation of ribosomal subunits containing intact rRNA from human placenta: estimation of functional activity of 80S ribosomes.

We have elaborated a method for the isolation of ribosomal subunits from fresh unfrozen human placenta containing intact rRNA and a complete set of ribosomal proteins. Activity of 80S ribosomes obtained by reassociation of 40S and 60S subunits in nonenzymatic poly(U)-dependent binding of Phe-tRNA(Phe) was equal to 80% (above 1.5 mol [14C]Phe-tRNA(Phe) is coupled to 1 mol of ribosomes). The activity of 80S ribosomes in poly(U)-directed synthesis of polyphenylalanine was tested in a polysome-free protein-synthesizing system from rabbit reticulocytes. About 100 mol of phenylalanine residue was polymerized by a mole of ribosomes at a rate of 0.83 residues per minute in this system (2 h, 37 degrees C).     

Preparation of Escherichia coli 70S ribosomes labeled with 35S

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.     

Phosphorylation of ribosomal proteins influences subunit association and translation of poly (U) in Streptomyces coelicolor

The occurrence of phosphorylated proteins in ribosomes of Streptomyces coelicolor was investigated. Little is known about which biological functions these posttranslational modifications might fulfil. A protein kinase associated with ribosomes phosphorylated six ribosomal proteins of the small subunit (S3, S4, S12, S13, S14 and S18) and seven ribosomal proteins of the large subunit (L2, L3, L7/L12, L16, L17, L23 and L27). The ribosomal proteins were phosphorylated mainly on the Ser/Thr residues. Phosphorylation of the ribosomal proteins influences ribosomal subunits association. Ribosomes with phosphorylated proteins were used to examine poly (U) translation activity. Phosphorylation induced about 50% decrease in polyphenylalanine synthesis. After preincubation of ribosomes with alkaline phosphatase the activity of ribosomes was greatly restored. Small differences were observed between phosphorylated and unphosphorylated ribosomes in the kinetic parameters of the binding of Phe-tRNA to the A-site of poly (U) programmed ribosomes, suggesting that the initial binding of Phe-tRNA is not significantly affected by phosphorylation. On contrary, the rate of peptidyl transferase was about two-fold lower than that in unphosphorylated ribosomes. The data presented demonstrate that phosphorylation of ribosomal proteins affects critical steps of protein synthesis.     

Mechanism of protein synthesis inhibition during stationary phase in Bacillus stearothermophil US

Summary The appearance of a protein (association factor I) in ribosomes from Bacillus stearothermophilus at stationary phase of growth is described. Association factor I is present on 30S subunits and 30S–50S ribosomal couples, but not on 50S subunits. This protein is responsible for the low levels of polyphenylalanine synthesis shown by stationary phase ribosomes. Association factor I is able to bind to free 30S–50S ribosomal couples but not to polysomes, and exerts its effect by inhibiting the initiation step of protein synthesis. Ribosomes preincubated with association factor I have a decreased ability for polypeptide snythesis directed phage mRNA or poly(U).     

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.     

Modification of Ribosomes in Cryptopleurine-Resistant Mutants of Yeast

Cryptopleurine-resistant mutants of Saccharomyces cerevisiae were isolated. A single, recessive nuclear gene, very closely linked to the mating locus (2.1 centimorgans), is responsible for resistance. Ribosomes from the mutants were found to be resistant to cryptopleurine when analyzed by poly(U)-directed polyphenylalanine synthesis. Analysis of the distribution of ribosomes between monosomes and polysomes in sensitive cells exposed to cryptopleurine suggests that some step is inhibited during the elongation phase of protein synthesis.     

Reassociation of cytoplasmic ribosomal subunits of barley and its virescens mutant to form active hybrids

Polyphenylalamine synthesis by cytoplasmic ribosomes of Gateway barley (Hordeum vulgare) and its virescens single gene nuclear mutant was compared. The cytoplasmic 80S ribosomes were isolated from unimbibed embryo material and the ribosomes were dissociated into their component 60S and 40S subunits by centrifugation through sucrose gradients containing high KCl-to-MgCl₂ buffer. These separated subunits could be reassociated by resuspension in buffer having about equimolar concentrations of MgCl₂ and KCl. Both homologous and heterologous combinations of the subunits reassociated to give monomeric 80S ribosomes, and the derived monomers as well as various combinations of the individual subunits showed equivalent activity in an in vitro system for poly (U)-directed polyphenylalanine synthesis.     

Inhibition of translation in eukaryotic systems by harringtonine.

The Cephalotaxus alkaloids harringtonine, homoharringtonine and isoharringtonine inhibit protein synthesis in eukaryotic cells. The alkaloids do not inhibit, in model systems, any of the steps of the initiation process but block poly(U)-directed polyphenylalanine synthesis as well as peptide bond formation in the fragment reaction assay, the sparsomycin-induced binding of (C)U-A-C-C-A-[3H]Leu-Ac, and the enzymic and the non-enzymic binding of Phe-tRNA to ribosomes. These results suggest that the Cephalotaxus alkaloids inhibit the elongation phase of translation by preventing substrate binding to the acceptor site on the 60-S ribosome subunit and therefore block aminoacyl-tRNA binding and peptide bond formation. However, the Cephalotaxus alkaloids do not inhibit polypeptide synthesis and peptidyl[3H]puromycin formation in polysomes. Furthermore, these alkaloids strongly inhibit [14C]trichlodermin binding to free ribosomes but hardly affect the interaction of the antibiotic with yeast polysomot interact with polysomes and therefore only inhibit cycles of elongation. This explains the polysome run off that has been observed by some workers in the presence of harringtonine.     

The A-site finger in 23 S rRNA acts as a functional attenuator for translocation

Helix 38 (H38) in 23 S rRNA, which is known as the "A-site finger (ASF)," is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.     

Stimulation of gastric mucosal protein synthesis by different molecular forms of gastrin.

Protein synthesis in gastric mucosa was studied by measuring the incorporation of labeled amino acids into protein by isolated gastric mucosal ribosomes in a cell-free system. In 48-hour fasted rats, administration of the synthetic analogues pentagastrin, tetragastrin and gastrin-17 or naturally occurring molecular forms of human gastrin (G-14, G-34) markedly enhanced (23-123%) the capacity of the gastric mucosal ribosomes to synthesize endogenous mRNA-directed protein in a cell-free system. In the presence of exogenous mRNA (poly-U), the gastric mucosal ribosomes from the saline-treated controls showed a higher poly(U)-directed protein synthesis, compared to each fo the gastrin-treated groups. The protein/polyphenylalanine ratio which represents a ratio of polysomes to monosomes was found increased in ribosomes from the gastrin-treated groups.     

Correlation of inhibition of ribosomal GTPase and protein synthesis by abrin and ricin.

The rate of inhibition of protein synthesis and ribosome-dependent GTPase activity by abrin and ricin was determined. Ribosomes were treated with toxin A-chains for increasing periods of time and they assayed for their ability to synthesize polyphenylalanine and hydrolyze GTP. The rate of inhibition of GTPase activity of the 60S subunit derived 8S ribonucleoprotein complex was also determined. Although the degree of maximal inhibition of protein synthesis and GTPase activity was different, the rates of inhibition were almost identical. The results support a conclusion that abrin and ricin inhibit protein synthesis by inactivating ribosome-dependent GTPase.     

Involvement of 30S ribosomal protein S1 in poly(U)-directed polyphenylalanine synthesis.

The effect of 30S ribosomal protein S1 on poly(U)-directed polyphenylalanine synthesis was studied using a highly purified cell-free system which was devoid of endogenous S1. The system consisted of homogeneous preparations of EF-Tu, EF-Ts, and EF-G, and 70S ribosomes from which protein S1 had been removed by poly(U)-cellulose column chromatography. It was found that protein S1 was indispensable for translation of poly(U) by an S1-depleted system at low concentrations of poly(U). On the other hand, at higher concentrations of poly(U), a considerable amount of polyphenylalanine was synthesized in the absence of added S1. The stimulatory effect of S1 was observed at all Mg2+ concentrations examined but was most pronounced at 10 mM Mg2+. Some physicochemical properties of the protein were also studied. It was demonstrated that the protein has an elongated shape with an axial ratio of approximately 8.5.