Analysis of proteinase A function in yeast

The antibiotic, micrococcin, binds to complexes formed between bacterial 23-S ribosomal RNA and ribosomal protein L11 and, in doing so, inhibits of thiostrepton. In assay systems simulating partial reaction of protein synthesis, micrococcin inhibits a number of processes believed to involve the ribosomal A site while stimulating GTP hydrolysis dependent upon ribosomes and elongation factor EF-G. The latter effect is not observed upon ribosomes lacking a protein homologous with protein L11. Nor is it apparent upon those containing 23-S RNA previously subjected to the action of a specific methylase known to render ribosomes resistant to thiostrepton. It is concluded that stimulation by micrococcin of factor-dependent GTP hydrolysis results from the binding of the drug to its normal target site which involves 23-S RNA and protein L11.     

Dual actions of viomycin on the ribosomal functions.

Viomycin was observed to inhibit poly[U]- or f2 RNA-directed protein synthesis in an $\text{E. coli}$ cell-free system. The former was more profoundly affected than the latter. Both initiation complex formation on the 30S ribosomal subunit and on 70S ribosomes were prevented by the antibiotic. In the peptide chain elongation process, viomycin did not significantly affect aminoacyl-tRNA binding to ribosomes and the peptidyl transferase reaction, but markedly inhibit translocation of peptidyl-tRNA from the acceptor site to the donor site. The mechanism of action of the drug appeared to be unique.     

Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes.

Hygromycin B is an unusual aminoglycoside antibiotic active against both prokaryotic and eukaryotic cells. Hygromycin B at 0.38 mM concentration completely halts yeast cell growth in rich media, presumably by preventing protein synthesis by cytoplasmic ribosomes. Polypeptide synthesis in cell-free extracts from rabbit reticulocytes, wheat germ and yeast is strongly blocked by low concentrations of hygromycin B. The antibiotic inhibits peptide chain elongation by yeast polysomes by preventing elongation factor EF-2-dependent translocation, although it does not affect either the formation of the EF-2-GTP-ribosome complex or the EF-2- and ribosome-dependent GTP hydrolysis which takes place uncoupled from translocation. The inhibition of translocation by hygromycin B might result from the stabilization of peptidyl-tRNA bound to the ribosomal acceptor site, since the stability of [3H]Phe-tRNA-EF-1-poly(U)-ribosome and [3H]Phe-tRNA-poly(U)-ribosome complexes is increased in the presence of hygromycin B. The inhibition of polyphenylalanine synthesis by reticulocyte ribosomes and enzymic translocation of peptidyl-tRNA by yeast polysomes can be reversed by increasing concentrations of EF-2 suggesting a relationship between the binding sites of EF-2 and hygromycin B on the ribosome. Neither non-enzymic translocation, that takes place in the presence of high potassium concentrations, nor the peptide bondforming step are affected by hygromycin B.     

Inhibition of translation in bacterial and eukaryotic systems by the antibiotic anthelmycin (hikizimycin).

Anthelmycin inhibits protein synthesis on both pro- and eukaryotic ribosomes by preventing the peptide bond-forming reaction. The drug is structurally similar to certain other 4-aminohexosyl cytosine antibiotics including blasticidin S, gougerotin, amicetin and bamicetin although unlike these compounds anthelmycin lacks an aminoacyl moiety. It is proposed that anthelmycin inhibits the ribosomal peptidyl transferase centre by associating with a site that overlaps the (related) ribosomal receptor site(s) for the other four inhibitors.     

Inhibition of protein synthesis by didemnin B: how EF-1alpha mediates inhibition of translocation

The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein synthesis in cells and in vitro. The stage at which DB inhibits protein synthesis in cells is not known, although dehydrodidemnin B arrests translation at the stage of polypeptide elongation. Inhibition of protein synthesis by DB in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models of protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1alpha; however, the dependence upon EF-1alpha was previously unexplained. It is shown here that DB binds only weakly to EF-1alpha/GTP in solution, but binds to ribosome. EF-1alpha complexes with a dissociation constant K(d) = 4 microM. Thus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated ADP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 binding to pre-translocative ribosome.EF-1alpha complexes, thus preventing ribosomal translocation. Based on this model for protein synthesis inhibition by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1alpha function in which this elongation factor does not fully depart the ribosome during polypeptide elongation.     

The tsr gene-coding plasmid pIJ702 prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans

Streptomyces lividans normally accumulated high levels of ppGpp during nutritional shift-down. Its accumulation was, however, severely inhibited when a small amount of thiopeptin (an analogue of thiostrepton) was included in the transfer medium. In contrast, a S. lividans strain, which harbours the plasmid pIJ702 carrying the tsr gene resist to thiopeptin through methylation of the 23S rRNA, was still capable of accumulating ppGpp in the presence of large amounts of thiopeptin. These results indicate that the rRNA methylation resulting from the action of tsr gene prevents thiopeptin not only from inhibiting cell-growth but also from inhibiting ppGpp synthesis. The results also indicate that the observed accumulation of ppGpp during nutritional shift-down was associated with ribosomal function, as has been shown in E. coli and B. subtilis.     

Trichodermin,a possible inhibitor of the termination process of protein synthesis

The mode of action of the antibiotic, trichodermin, on yeast cells has been investigated. Trichodermin specifically inhibits protein synthesis and, during the in vivo inhibition of protein synthesis, ribosomes remain in polyribosomes rather than shifting to monoribosomes. This observation suggests that trichodermin inhibits either an elongation step or a termination step of protein biosynthesis. These two possibilities were distinguished by comparing the action of trichodermin with that of cycloheximide, a known elongation inhibitor, upon the reformation of polyribosomes during recovery from a block in polypeptide chain initiation. Cycloheximide slows the recovery of polyribosomes from monoribosomes following a block in polypeptide chain initiation whereas trichodermin enhances the recovery of polyribosomes. This observation is interpreted to mean that trichodermin primarily inhibits the termination step of protein biosynthesis.     

Inhibition by thiopeptin of ribosomal functions associated with T and G factors

Thiopeptin, a new antibiotic, inhibits T factor-dependent binding of phe-tRNA to ribosomes, without appreciable inhibition of GTP-Tu-phe-tRNA complex formation. GTP hydrolysis coupled with the phe-tRNA binding is affected by the antibiotic. It also interferes with fusidic acid-sensitive hydrolysis of GTP caused by interaction with G factor and ribosomes. Siomycin acts similarly.     

Nonenzymic translocation and spontaneous release of noncognate peptidyl transfer ribonucleic acid from Escherichia coli ribosomes

Poly(uridylic acid)-programmed ribosomes have been used to synthesize the noncognate peptidyl-tRNA Ac-Phe-Tyr-tRNATyr and its cognate counterpart Ac(Phe)2-tRNAPhe. After synthesis, Ac(Phe)2-tRNAPhe remains, as expected, in the ribosomal acceptor (A) site, but the noncognate AcPhe-Tyr-tRNATyr does not; part of it spontaneously falls off the ribosome and the rest translocates, without elongation factor (EF) G, to the ribosomal donor site. The inhibitor of translocation viomycin prevents both the spontaneous release and the nonenzymatic translocation by confining the noncognate peptidyl-tRNA to the A site. Under these conditions, the interaction of AcPhe-Tyr-tRNATyr with the A site appears to be similar to that of Ac(Phe)2-tRNAPhe without the antibiotic, and EF-G promotes the translocation and subsequent elongation of both peptidyl-tRNAs to comparable extents. The results indicate that, without viomycin, the noncognate peptidyl-tRNA is weakly held in the ribosomal A site and support the proposal that the release of peptidyl-tRNA occurring during protein synthesis in vivo is related to a ribosomal editing mechanism which discards mistranslated nascent proteins [Menninger, J. R. (1977) Mech. Ageing Dev. 6, 131].     

Antibiotics and compounds affecting translation by eukaryotic ribosomes. Specific enhancement of aminoacyl-tRNA binding by methylxanthines

Summary The mode and site of action of inhibitors of translation (initiation, elongation and termination of protein synthesis) in eukaryotic systems is reviewed. The isolation and characterization of a factor is described that binds Ac-Phe-tRNA to form a complex made up of binding factor, Ac-Phe-tRNA, and ribosome. The binding of Ac-Phe-tRNA probably occurs at the ribosomal site involved in the binding of the initiator substrate Met-tRNA_F. The effect of inhibitors of the initiation phase of protein synthesis on the nonenzymic Ac-Phe-tRNA binding to ribosomes is investigated. The two sites translocation model for translation in eukaryotic cells is presented and the effects of inhibitors on the various steps of protein synthesis are determined empirically. The site of action of inhibitors of peptide bond formation at the ribosomal peptidyl transferase center is elucidated. The action of inhibitors of translocation is studied in model cell-free systems from human cells. In addition, a number of methylxanthines are shown to enhance the elongation phase in polypeptide synthesis by stimulating the enzymic binding of aminoacyl-tRNA. The effect of caffeine, theophylline and its derivatives are shown to be fairly specific and dependent on the ribosome concentration. Aminophylline is shown to have a similar effect but also enhances aminoacyl-tRNA synthetase activity at low Mg^{+ +} concentrations, probably by displacing the optimal concentration of Mg^{+ +} in the reaction. This second effect of aminophylline appears to be due to the ethylenediamine moiety of aminophylline since it is also observed in the presence of different polyamines but not in the presence of caffeine or theophylline.An invited article.     

The mode of action of alpha sarcin and a novel assay of the puromycin reaction.

A new technique was developed for measuring the amount of peptidyl-tRNA in a protein-synthesizing system in vitro. By this technique the course of the puromycin reaction may be followed and the modes of action of various inhibitors of protein synthesis readily determined. We conclude that the polypeptide alpha sarcin inhibits the binding of aminoacyl-tRNA into the ribosomal 'A' site, that sparsomycin inhibits the peptidyl transferase reaction and that cycloheximide may block translocation.     

Conformational changes of L-rRNA during elongation of polypeptide

A ribosome undergoes significant conformational changes during elongation of a polypeptide chain, and these are correlated with structural changes of rRNAs. We tested 15 different oligonucleotides complementary to the selected, highly conserved seqences of rRNAs (L-rRNA, 5S rRNA and tRNA) important in protein biosynthesis. We carried out a reaction of binding Phe-tRNA to A site and a polymerization of polypeptide chains on the ribosomes converted either to pre- or to posttranslocational states. The inhibition of polymerization reaction by complementary oligonucleotides was high in all ribosomal states. The efficiency of inhibition of binding reaction was lower and more diverse than was the polypeptide elongation. We conclude that the selected oligonucleotides inhibit polypeptide synthesis with different effectivity, primarily depending on L-rRNA conformation within ribosome architecture.     

Effect of Purine and Pyrimidine Analogues on Growth and RNA Metabolism in the Soybean Hypocotyl-the Selective Action of 5-Fluorouracil

The effects of several base analogues and cycloheximide on RNA synthesis, protein synthesis, and cell elongation were studied in excised soybean hypocotyl. None of the pyrimidine analogues tested affected growth or protein synthesis; only 5-fluorouracil appreciably inhibited RNA synthesis. 8-Azaguanine and 6-methylpurine markedly inhibited RNA and protein synthesis and cell elongation. Cycloheximide effectively inhibited both cell elongation and protein synthesis.The results show that 5-fluorouracil selectively inhibited ribosomal and soluble RNA synthesis without affecting the synthesis of D-RNA. These results indicate that the requirement for RNA synthesis to support continued protein synthesis and cell elongation is restricted to the synthesis of D-RNA.5-Fluorouracil was incorporated into all classes of RNA in a form believed to be 5-fluorouridylic acid.Cycloheximide markedly inhibited the accumulation of ribosomal RNA, but the results indicate that CH did not inhibit, per se, the synthesis of ribosomal RNA. The accumulation of newly synthesized D-RNA was only slightly affected by cycloheximide. These results show that the inhibition of cell elongation by cycloheximide correlates with the inhibition of protein synthesis, but not with the effect on RNA metabolism.     

蛋白质生物合成的第四步：翻译终止后复合物的解体

蛋白质合成过程一般被归纳为由合成的起始、肽链的延伸和合成的终止组成的三步曲 . 然而,随着对核糖体再循环因子 (ribosome recycling factor , RRF) 在蛋白质合成过程中作用的深入研究,人们提出了蛋白质生物合成应是四步曲, 这第四步就是翻译终止后核糖体复合物的解体 , 也就是通常说的核糖体循环再利用 . 简要地介绍了翻译终止后复合物解体的可能机制：核糖体再循环因子和蛋白质合成延伸因子 G 在核糖体上协同作用催化这一过程的完成 .     

Protein toxin inhibitors of protein synthesis.

Two classes of extremely toxic proteins kill eukaryotic cells by covalently modifying unique structural features of components that are essential for protein synthesis. Intoxication by these proteins results from the entry of a catalytic fragment into the cytoplasm. One class is typified by diphtheria toxin and Pseudomonas exotoxin A. The catalytic component of these toxins ADP-ribosylates and inactivates elongation factor 2 which is an essential participant in protein synthesis. This modification occurs at a unique post-translational histidine derivative, diphthamide, that is present in the ribosomal binding site of the elongation factor. The two toxins differ in their molecular organization but appear to possess identical reaction mechanisms and very similar active sites. The other class contains two types of toxins typified, respectively, by alpha-sarcin, a member of a family of fungal toxins, and ricin, a member of a group of closely related plant proteins collectively termed ribosome-inactivating proteins. The catalytic components of the two types of toxins in this second class inactivate the large ribosomal subunit through two different hydrolytic alterations of 23-28S RNA. alpha-Sarcin and its congeners act as a specific endonuclease whereas ricin and its congeners act as a specific N-glycosidase. These hydrolytic cleavages occur at a pair of adjacent nucleotides within a highly conserved sequence near the 3' terminus of 23-28S RNA. The covalent integrity of this region of RNA is essential to elongation factor-dependent ribosomal functions and is located within the ribosomal binding domain of these factors. Both of these classes of toxins are being employed as 'magic bullets' to eliminate pathological cells. By combining the catalytic component of these toxins with various cell targeting components, useful and specific anticancer and immunomodulatory agents have been created.     

RALyase; a terminator of elongation function of depurinated ribosomes

Plant ribosomal RNA apurinic site specific lyase (RALyase) cleaves the phosphodiester bond at the depurinated site produced by ribosome-inactivating protein, while the biological role of this enzyme is not clear. As the depurinated ribosomes retain weak translation elongation activities, it was suggested that RALyase completes the ribosome inactivation. To confirm this point, we measured the effects of the phosphodiester cleavage using a fusion of wheat RALyase produced with a cell-free protein synthesis system from wheat germ. The results indicated that RALyase diminishes the residual elongation activities of the depurinated ribosomes.     

Ribosomal RNA and protein mutants resistant to spectinomycin.

We have compared the influence of spectinomycin (Spc) on individual partial reactions during the elongation phase of translation in vitro by wild-type and mutant ribosomes. The data show that the antibiotic specifically inhibits the elongation factor G (EF-G) cycle supported by wild-type ribosomes. In addition, we have reproduced the in vivo Spc resistant phenotype of relevant ribosome mutants in our in vitro translation system. In particular, three mutants with alterations at position 1192 in 16S rRNA as well as an rpsE mutant with an alteration of protein S5 were analysed. All of these ribosomal mutants confer a degree of Spc resistance for the EF-G cycle in vitro that is correlated with the degree of growth rate resistance to the antibiotic in culture.     

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.     

The chloramphenicol receptor site in Escherichia coli in Vivo affinity labeling by monoiodoamphenicol

Monoiodoamphenicol inhibits protein synthesis in exponentially growing Escherichia coli without measurable delay. The inhibition follows first-order kinetics suggesting that one molecule of monoiodoamphenicol is sufficient to inhibit one ribosome. However, only 60% of the ribosomes were susceptible to irreversible inhibition by monoiodoamphenicol. The residual 40% of the rate of protein synthesis after monoiodoamphenicol treatment was not significantly different, when the time of treatment was increased up to six hours or the drug concentration was raised to 1000 μg/ml.Analysis of the monoiodoamphenicol-labeled cell proteins by various uni- and two-dimensional gel electrophoreses and by sucrose gradient centrifugation shows that the cell target of the covalent labeling reaction of monoiodoamphenicol is almost exclusively the ribosome. The amount of label incorporated into ribosomal protein is proportional to the degree of inhibition of protein synthesis by monoiodoamphenicol. However, more than one ribosomal protein reacted. The predominantly labeled proteins are S6, L16 and L24. It is proposed that all three proteins are associated with the receptor site of monoiodoamphenicol.