Inhibition by ricin of protein synthesis in vitro. Ribosomes as the target of the toxin

1. Ricin (a toxic protein from the seeds of Ricinus communis) is a powerful inhibitor of the poly(U)-directed incorporation of phenylalanine into polypeptides catalysed by isolated rat liver ribosomes and elongation factors 1 and 2 (EF 1 and EF 2). The inhibition can be largely overcome by increasing the concentration of ribosomes. 2. The toxin does not affect the binding of phenylalanyl-tRNA to ribosomes catalysed by EF 1, nor does it inhibit the puromycin reaction used as a test for peptide-bond formation catalysed by ribosomes. 3. Ricin inhibits the ribosome-linked GTP hydrolysis catalysed by EF 2. 4. Ribosomes treated with ricin and washed through sucrose gradients containing 0.6m-NH(4)Cl are functionally inactive in those assay systems that are sensitive to the presence of added toxin. 5. It is suggested that ricin brings about an irreversible modification of ribosomes which impairs their ability to interact with EF 2. Since ricin inhibits at a molar concentration much lower than that of ribosomes it probably acts catalytically. No added cofactor is necessary for the inhibitory action of the toxin.     

Studies on the binding of bacterial elongation factors EF Tu and EF G to ribosomes

When EF G² from Escherichia coli or Pseudomonas fluorescens is pre-bound to ribosomes in the presence of GMD, or GTP and fusidic acid, a differential effect is observed on the subsequent EF Tu-catalyzed binding of aminoacyl-tRNA to ribosomes. The EF G from E. coli nearly completely prevents the binding reaction, whereas the corresponding factor from P. fluorescens displays a significantly lower inhibitory effect. Both EF G factors form stable complexes with ribosomes and are equally efficient in the polymerization reaction. The difference in inhibitory properties between the two factors persists over a wide range of NH₄Cl concentration.     

Effect of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 on translocation.

1. The effect of elongation factor 2 (EF 2) and of adenosine diphosphate-ribosylated elongation factor 2 (ADP-ribosyl-EF 2) on the shift of endogenous peptidyl-tRNA from the A to the P site of rat liver ribosomes (measured by the peptidyl-puromycin reaction) and on the release of deacylated tRNA (measured by aminoacylation) was investigated. 2. Limiting amounts of EF2, pre-bound or added to ribosomes, catalyse the shift of peptidyl-tRNA in the presence of GPT; when the enzyme is added in substrate amounts GMP-P(CH2)P [guanosine (beta, gamma-methylene)triphosphate] can partially replace GTP. ADP-ribosyl-EF 2 has no effect on the shift of peptidyl-tRNA when present in catalytic amounts, but becomes almost as effective as EF 2 when added in substrate amounts together with GTP; GMP-P(CH2)P cannot replace GTP. 3. The release of deacylated tRNA is induced only by substrate amounts of added EF 2 and also occurs in the absence of guanine nucleotides. In this reaction ADP-ribosyl-EF 2 is only 25% as effective as EF 2 in the absence of added nucleotide, but becomes 60-80% as effective in the presence of GTP or GMP-P(CH2)P. 4.The results obtained on protein-synthesizing systems are consistent with the hypothesis that ADP-ribosyl-EF 2 can operate a single round of translocation followed by binding of aminoacyl-tRNA and peptide-bond formation. 5. From the data obtained with the native enzyme it is concluded that the two moments of translocation require different conditions of interaction of EF 2 with ribosomes; it is suggested that the shift of peptidyl-tRNA is catalysed by EF 2 pre-bound to ribosomes, and that the release of tRNA is induced by a second molecule of interacting EF 2. The hydrolysis of GTP would be required for the release of pre-bound EF 2 from ribosomes. 5. The inhibition of the utilization of limiting amounts of EF 2 on ADP-ribosylation is very likely the consequence of a concomitant decrease in the rate of association and dissociation of the enzyme from ribosomes.     

Studies on elongation factor II from calf brain

Elongation factor II (EF2) has been purified from calf brain, and its reactions with guanosine nucleotides and ribosomes have been studied. Its behavior is, in general, similar to that observed with EF2 from other eukaryote sources. Thus, in the presence of GTP or GDP, EF2 interacts with ribosomes to form a ribosome-EF2-GDP complex. Fusidic acid has little effect on the stability of this complex, which suggests that it is more stable than the corresponding complex from prokaryote systems. As assayed by a nitrocellulose filter technique, only GTP, GDP, dGTP and GDPCP are bound to ribosomes dependent on EF2. In the absence of ribosomes, an EF2-GTP or EF2-GDP complex can be detected. Fusidic acid at relatively high concentrations inhibits their formation, but diphtheria toxin in the presence of NAD does not. The EF2-GTP complex has been separated from unbound GTP by gel filtration, and the reactivity of the complex with ribosomes has been investigated. When EF2-GTP is incubated with ribosomes, GTP hydrolysis occurs, and evidence for a ribosome-EF2-GDP complex has been obtained. The results thus suggest that the EF2-GTP complex may be an intermediate in the binding of EF2 to ribosomes. Based on molecular sieve chromatography, it appears that the stability of these complexes is ribosome-EF2-GDP > EF2-GTP > EF2-GDP.     

Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin.

The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared. Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene)-diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only. EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not. The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins. The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin. The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them. EF2-dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin. In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin. The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation. In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes.     

Effect of modeccin on the steps of peptide-chain elongation.

Modeccin inhibits polypeptide-chain elongation catalysed by Artemia salina (brine shrimp) ribosomes by inactivating the 60 S ribosomal subunit. Among the individual steps of elongation, peptide-bond formation, catalysed by 60 S peptidyltransferase, is unaffected by the toxin, whereas the binding of EF 2 (elongation factor 2) to ribosomes is strongly inhibited. Modeccin does not affect the poly(U)-dependent non-enzymic binding of either deacylated tRNAPhe or phenylalanyl-tRNA to ribosomes. The inhibitory effect of modeccin on the EF 1 (elongation factor 1)-dependent binding of phenylalanyl-tRNA is discussed, since it is decreased by tRNAPhe, which stimulates the binding reaction. The analysis of the distribution of ribosome-bound radioactivity during protein synthesis shows that modeccin consistently inhibits the radioactivity bound as long-chain peptides, but depending on the experimental conditions, can leave unchanged or even greatly stimulates the radioactivity bound as phenylalanyl-tRNA and/or short-chain peptides. It is concluded that, during the complete elongation cycle, modeccin does not affect the binding of the first aminoacyl-tRNA to ribosomes, but inhibits some step in the subsequent repetitive activity of either EF 1 or EF 2. The results obtained indicate that the mechanism of action of modeccin is very similar to that of ricin and related plant toxins such as abrin and crotin.     

Effect of alpha-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes.

alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system.     

Evolutionary divergence of an elongation factor 3 from Cryptococcus neoformans

Elongation factor 3 (EF3) is considered a promising drug target for the control of fungal diseases because of its requirement for protein synthesis and survival of fungi and a lack of EF3 in the mammalian host. However, EF3 has been characterized only in ascomycete yeast. In order to understand the role of EF3 in a basidiomycete yeast, we cloned the gene encoding EF3 from Cryptococcus neoformans (CnEF3), an important fungal pathogen in immunocompromised patients, including those infected with human immunodeficiency virus. CnEF3 was found to encode a 1,055-amino-acid protein and has 44% identity with EF3 from Saccharomyces cerevisiae (YEF3). Expressed CnEF3 exhibited ATPase activity that was only modestly stimulated by ribosomes from S. cerevisiae. In contrast, CnEF3 showed tight binding to cryptococcal ribosomes, as shown by an inability to be removed under conditions which successfully remove Saccharomyces EF3 from ribosomes (0.5 M KCl or 2 M LiCl). CnEF3 also poorly complemented a YEF3 defect in a diploid null mutant and two temperature-sensitive mutants which have been shown previously to be complemented well by EF3 from other ascomycetes, such as Candida albicans. These data clearly identify the presence of a functioning EF3 in the basidiomycete yeast C. neoformans, which demonstrates an evolutionary divergence from EF3 of ascomycete yeast.     

Subcellular distribution and functional properties of different forms of elongation fractor EF1 from wheat embryos.

Elongation factor EF1 was found in a low salt homogenate of wheat embryos, either in the 100 000 X g supernatant or in the ribosome pellet. The ribosome-linked EF1 (EF1R), deteched by high salt washing, was purified to electrophoretical homogenetiy and its molecular and functional properties compared to those of a purified high molecular weight species of EF1 obtained from cytoplasm (EF1H). The two forms are associations of different polypeptides having in common only the polypeptide which can form the ternary complex with aminoacyl-tRNA and GTP. Whereas EF1R is able to fulfill all the EF1 functions, EF1H, incubated with ribosomes completely deprived of elongation factors, can catalyze the aminoacyl-tRNA binding to ribosomes, but, in the presence of EF2, forms only a very small amount of poly(Phe).     

Available sulphydryl groups of mammalian ribosomes in different functional states

The numbers of sulphydryl groups on NH₄Cl-washed rat liver polyribosomes in different functional states were measured under carefully standardized conditions with ¹⁴C-labelled N-ethylmaleimide and ³⁵S-labelled 5,5-dithio-bis(2-nitrobenzoic acid). Ribosomes denatured with urea had 120 titratable sulphydryl groups, 60 on each subunit, whereas native ribosomes invariably showed fewer available sulphydryl groups. Ribosomes stripped of transfer RNA (S-type ribosomes) had 55 available sulphydryl groups. Ribosomes bearing the growing peptidyl-tRNA at the acceptor site had 41 sulphydryl groups available. If these A-type ribosomes were labelled with ¹⁴C-labelled N-ethylmaleimide and dissociated into subunits, 23 of the labelled sulphydryl groups were found on the 60 S subunit and 19 on the 40 S subunit. After translocation of the peptidyl-tRNA to the donor position on ribosomes (D ribosomes), the number of available sulphydryl groups increased to 72, of which 43 were on the 60 S subunit and 29 on the 40 S subunit. This demonstrates that both subunits participate in the change of peptidyl-tRNA from the A to D positions. When the D ribosomes were reacted with EF2 (elongation factor) and GTP, the available sulphydryl groups increased to 82; addition of EF2 alone or with GDP, GDPCP or ATP failed to cause this increase, which has accordingly been attributed to an energy-dependent conformational change in the ribosome.Ribosomes were reconstructed from subunits with poly(U) and Phe-tRNA. In the presence of poly(U) only, a ribosome with 55 available SH groups was formed, thus corresponding to the stripped ribosomes. When both poly(U) and Phe-tRNA were present, a ribosome was formed with 44 available sulphydryl groups, corresponding approximately to an A-type ribosome. Since no EF1 or GTP was used in reconstructing this ribosome, these data indicate that the conformation of A-type ribosomes is not dependent on EF1 or GTP, but is due to the presence of tRNA at the acceptor site.We therefore incline to the view that the observed changes in available SH groups reflect conformational changes, with an opening up of ribosome structure as it progresses from having the peptidyl-tRNA at the A position to the D position and then binds EF2 and GTP, followed by a restoration of the more compact from when the incoming aminoacyl-tRNA is then bound.     

On the mechanism of a calcium-associated defect of oxidative phosphorylation in progessive muscular dystrophy

Partially purified elongation factor 1 preparations from calf brain, sheep brain, calf liver, and rabbit reticulocytes have been compared in their ability to interact with GTP and Phe-tRNA. A nitrocellulose filter assay has been used to study these interactions, and with all the EF1 preparations studied, evidence has been obtained for the formation of a Phe-tRNA·-EF1·-GTP complex. The ternary complex reacts with calf brain ribosomes in the presence of poly(U) resulting in a rapid hydrolysis of GTP and the binding of Phe-tRNA to the ribosome. Indirect evidence indicates that EF1·GDP is a product of this reaction. In the absence of poly(U) the intact complex reacts with the ribosomes without hydrolysis of GTP. The stability of the ternary complex was different with the various EF1 preparations, but the most stable complexes were prepared with calf brain EF1. Sephadex chromatography of the ternary complex shows that it contains a low molecular-weight species of the enzyme.     

Elongation factor 2 from Artemia salina embryos and its affinity for ribosomes

Crude extracts from Artemia salina undeveloped embryos do not contain detectable elongation-factor-2 (EF2) kinase and endogenous ADP-ribosylating activities. Accordingly, EF2 purified from this source is an enzyme relatively free from phosphorylated and ADP-ribosylated forms. Endogenous ADP-ribosyltransferase activity appears only after purification of EF2. The affinities of EF2 and of ADP-ribosyl-EF2 for ribosomes from A. salina undeveloped embryos have been calculated by measuring the ability of the factors to inhibit the N-glycosidase activity of ricin on ribosomes.     

Ribosome-bound elongation factor 2 escapes phosphorylation by Ca2+/calmodulin-dependent protein kinase III.

The activity of eukaryotic elongation factor 2 is regulated by phosphorylation catalysed by a highly specific Ca2+/calmodulin-dependent protein kinase. Phosphorylated EF2 binds to ribosomes with decreased affinity. The present evidence indicates that EF2 prebound to ribosomes is protected from phosphorylation, just as earlier evidence indicated that ribosome-bound EF2 is protected from ADP-ribosylation catalysed by diphtheria toxin. Ribosome-inactivating proteins ricin and gelonin, by interfering with the EF2-ribosome interaction, allow full phosphorylation of EF2.     

A novel reaction of reticulocyte peptide-chain elongation factor, EF2, with guanosine nucleotides

The formation of phenylalanyl puromycin from phenylalanyl-tRNA, bound nonenzymically or enzymically to reticulocyte ribosomes, requires the peptide-chain elongation factor, EF2², and GTP. However the GTP analogue, GDPCP, may replace GTP to a significant extent in this reaction. Other purine or pyrimidine nucleotides have little or no activity. Multistep experiments with either GTP or GDPCP indicate that binding of EF2 to the ribosome for subsequent peptide formation may be a portion of the activity of the EF2 (independent of the translocation reaction) during the elongation process. Neomycin inhibits the formation of phenylalanyl puromycin using either GTP or GDPCP in this system.     

Inhibition by ricin of protein synthesis in vitro. Inhibition of the binding of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 to ribosomes.

The binding of EF2 (elongation factor 2) and of ADP-ribosyl-EF 2 to rat liver ribosomes is inhibited by ricin. This result suggests that the native enzyme and its ADP-ribose derivative have the same or closely related binding sites on the ribosome. The inhibition by ricin of the binding of EF 2 to ribosomes is consistent with the previous observation that ricin affects EF 2-catalysed translocation during polypeptide chain elongation.     

Amino acid incorporation in a cell-free system derived from rat liver studied with the aid of selenodiglutathione

Selenodiglutathione (GSSeSG), a potent inhibitor of elongation factor 2 (EF2) has been used to study amino acid incorporation in a rat liver cell-free system. While translocation of the ribosomes was inhibited by GSSeSG, ribosomes with a free acceptor site were still capable of incorporating one amino acid residue. From this the average number of amino acids incorporated per ribosomes was calculated to be 2--5. In this respect virtually no difference has been observed between ribosomes present on small or large aggregates. The time required for one translocation by all active ribosomes, and the time required for the incorporation of one amino acid (starting with aminoacyl-tRNA or amino acids) has also been determined. By incubation under conditions for amino acid incorporation, part of the ribosomes were completely inactivated whereas the rest remained as active as at the start of the incubation.     

Interaction of elongation factor 2 from wheat germ with guanosine nucleotides and ribosomes.

1. The amino acid composition of wheat germ EF2 differs to some extent from that of elongation factors from mammals and bacteria. 2. The purified wheat germ EF2, similarly as the factors from other sources, is active in the: EF1-dependent polymerization of phenylalanine; ribosome-dependent GTP hydrolysis; binding of guanosine nucleotides; and ADP-ribosylation in the presence of diphtheria toxin. Fusidic acid at a concentration of 1 mM inhibits all these EF2-dependent reactions. 3. Diphtheria toxin in the presence of NAD+ inhibits polymerization of phenylalanine but does not effect GTP binding to EF2. 4. Binding of GDP to wheat germ EF2 is inhibited by ribosomes. During interaction with ribosomes, GTP in EF2-GTP complex is rapidly hydrolysed to GDP. Both GTP and 5'-guanylmethylenediphosphonate competitively inhibit formation of the ribosome-EF2-GDP complex due to the replacement of GDP from the complex. The latter is stabilized by fusidic acid.     

The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome

The ribosomal elongation cycle describes a series of reactions prolonging the nascent polypeptide chain by one amino acid and driven by two universal elongation factors termed EF-Tu and EF-G in bacteria. Here we demonstrate that the extremely conserved LepA protein, present in all bacteria and mitochondria, is a third elongation factor required for accurate and efficient protein synthesis. LepA has the unique function of back-translocating posttranslocational ribosomes, and the results suggest that it recognizes ribosomes after a defective translocation reaction and induces a back-translocation, thus giving EF-G a second chance to translocate the tRNAs correctly. We suggest renaming LepA as elongation factor 4 (EF4).     

Chloroplast membrane biogenesis in chlamydomonas: Correlation between the formation of membrane components and membrane structure

The y-1 mutant of Chlamydomonas reinhardi, when allowed to green in the presence of chloramphenicol (CAP), an inhibitor of protein synthesis on 70s ribosomes, form photosynthetic membranes which contain somewhat less chlorophyll than those of cells greened in the absence of the drug. Photosystem I and II activities are drastically reduced in the CAP-greened cells, and specific alterations in the polypeptide composition of the thylakoid membranes are also observed. We have examined the internal structure of the thylakoid membranes from cells greened in the presence and absence of CAP, and have found that the large particles observed on the exoplasmic fracture face (EF) are substantially reduced in size and number in the CAP-greened cells. This structural defect seems related to the absence of significant photosystem activities in the CAP-greened cells, despite the presence, of most major membrane polypeptides. We suggest that CAP treatment results in a failure of the cell to organize functional reaction complexes, and is structurally reflected in the absence of large (EF) particles in such membranes. This defect can be repaired by allowing the affected cells to re-green in the absence of the drug, and the large particles reappear, paralleling an increase in photosynthetic activity.     

Factor-independent interaction of met-tRNA i and ApUpG with 40 S subunits of rat liver ribosomes

The small (40 S) subunit of rat liver ribosomes is capable of binding the initiator tRNA (Met-tRNA_i), in the absence of added protein factors, in marked preference to other aminoacyl-tRNAs. This binding requires magnesium ions, is codon (ApUpG)-specific, is not obtained with 60 S subunits, and is significantly higher than that observed with 80 S ribosomes. The 40 S subunit also exhibits a preference for ApUpG over several other trinucleotides. The reaction is inhibited by 60 S particles; it is also inhibited by compounds that effect chain initiation such as edeine and aurintricarboxylic acid, but not by cycloheximide, tetracycline or KF. All other aminoacyl-tRNAs, including Met-tRNA_m, bind more efficiently to 80 S ribosomes at low MgCl₂ concentrations with EF1 or in high Mg⁺⁺-containing solutions.