(3H)anisomycin binding to eukaryotic ribosomes

Anisomycin, a well-known inhibitor of eukaryotic ribosomes' peptidyl-transferase activity, specifically binds to the 60 S ribosome subunit. Quantitative studies on [³H]anisomycin binding to yeast and human tonsil ribosomes have shown that a maximum of one molecule of the antibiotic is bound per ribosome in both cases. There is a single type of binding to 60 S subunits but ribosomes themselves are not homogeneous with respect to [³H]anisomycin binding, since the interaction between antibiotic and ribosome occurs with two different affinities. Only ribosomes having the higher type of affinity for [³H]anisomycin are active in catalysing peptide bond formation, as tested in both the puromycin and the fragment reaction assays. Affinity of [³H]anisomycin for ribosomes is higher at 0 °C than at 30 °C. Affinity is decreased in the presence of ethanol.The acetate group in the 3′ position of the pyrrolidine ring of anisomycin is important for the anisomycin—ribosome interaction since deacetylanisomycin appears to have a mode of action similar to anisomycin but has an affinity for the ribosome that is 350 times smaller.The effect of certain peptidyl-transferase inhibitors has been tested on [³H]anisomycin binding to ribosomes. Using either yeast or human tonsil ribosomes a number of sesquiterpene antibiotics of the trichodermin group (trichodermin, trichodennol, fusarenon X and trichothecin) totally block [³H]anisomycin binding whereas puromycin and verrucarin A only partially inhibit the [³H]anisomycin interaction with ribosomes. Gougerotin, blasticidin S and actinobolin have no effect. Tenuazonic acid and sparsomycin inhibit [³H]anisomycin binding to ribosomes but the degree of inhibition differs between yeast and human tonsil ribosomes.     

The preparation of stereospecific tritium-labeled reduced nicotinamide adenine dinucleotide phosphate

A method has been developed for the preparation of a high specific activity stereospecifically labeled tritiated NADPH. In this procedure, tritium is enzymatically transferred from d-isocitric acid-2-³H (8 Ci/mmole) to the A face of a pyridine nucleotide during its stereospecific reduction, resulting in the formation of NADPH-4A-³H (2 Ci/mmole).     

THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to ¹⁴C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-³H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes.     

Comparison by photoaffinity labeling of the proteins involved in GTP hydrolysis from 70S ribosomes and a 5S RNA-protein complex from Bacillus stearothermophilus.

Photoaffinity labeling of 70S ribosomes from B. stearothermophilus by [³H]-1-(4-azidophenyl)-2-(5′-guanyl) pyrophosphate (APh-GDP) in the presence of fusidate and elongation factor G (EF-G) results in incorporation of tritium in the 50S proteins BL2, BL10 and BL22. Irradiation of the corresponding 5S RNA-protein complex in the presence of the GDP derivative gives only incorporation of tritium in BL10 and BL22. The proteins BL10 and BL22 comigrate in two dimensional gel electrophoresis with the 50S ribosomal proteins EL11 and EL18 from E. coli. The result suggests that the region at or near the guanine nucleotide binding site of the ribosome and the complex are the same. Since previous work has shown that the latter two are labeled upon irradiation of the ribosome with [³H]-APh-GDP, it is concluded that ribosomes from E. coli and B. stearothermophilus have structurally related GTPase sites.     

Comparative labelling of α1-acid glycoprotein by (3H)-borohydride or (125I)-iodide for use in a radioimmunoassay

The labelling of α₁-acid glycoprotein (AGP) with (³H)-sodium borohydride was compared to the labelling with (¹²⁵I)-sodium iodide by the chloramine T method in view to its use in a radioimmunoassay. The tritium labelling allowed to reach a high specific radioactivity similar to that obtained with iodide ((³H)-AGP: 29.8 mCi/mg; (¹²⁵I)-AGP: 30.5 mCi/mg). Each mole of sialic acid residue of AGP contains one atom of tritium. The stability of (³H)-AGP was better than that of (¹²⁵I)-AGP as indicated by its immunoreactivity as a function of time. Immunoreactivities and standard curves were similar for the two tracers but affinity of antiserum was higher for (¹²⁵I)-AGP than for (³H)-AGP. Tritium labelling by (³H)-borohydride will be very useful for glycoprotein antigens which cannot be labelled with (¹²⁵I)-iodide.     

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.     

Ribosome changes during translation

Studies on the quantitative binding of [³H]anisomycin are useful in determining conformational and/or structural changes on eukaryotic ribosomes. We have shown that yeast ribosomes have different structures depending on their functional states during the ribosome cycle as defined by their affinity for [³H]anisomycin.Free ribosomes, either in vivo run-off ribosomes (1 mm-sodium azide treatment or 8 °C incubation of spheroplasts) or puromycin-dependent released ribosomes, have an affinity defined by K_d = 3.3 to 3.6 μm.Ribosomes forming polysomes engaged in protein synthesis have at least two new different conformations (defined by K_d,H = 0.81 μm and K_d,L = 12 μm). These conformations have been ascribed to the pre and post-translocated steps of the elongation cycle in protein synthesis by blocking the polysomes with specific inhibitors of translation. Pre-translocated polysomes (polysomes blocked with cycloheximide) have an affinity of K_d^CHX = 12 μm and post-translocated polysomes (polysomes blocked with doxycycline) have an affinity of K_d^DC = 0.82 μm. These dissociation constants are identical to K_d,L and K_d,H obtained with control untreated polysomes, respectively.Moreover, a new ribosome conformation defined by K_d^DT = 1.5 μm and K_d^FA = 1.8 μm was found, by blocking the polysomes with the elongation factor, EF-2, bound by using either diphtheria toxin or fusidic acid.We also present evidence of the previously reported heterogeneity of standard preparations of eukaryotic ribosomes (Barbacid & Vazquez, 1974a) being a direct consequence of the high-salt washing treatment of ribosomes.     

Some biochemical properties of an acido-thermophilic archaebacterium,Sulfolobus acidocaldarius

To elucidate the phylogenic status of archaebacteria, some basic cellular components of an acido-thermophilic archaebacterium,Sulfolobus acidocaldarius, were studied. Poly(A) containing RNA was present in the cells, and performed the role of mRNA in a cell-free extract of reticulocyte or the archaebacteria. Poly(A) containing RNA was also found in other archaebacterial cells. The absence of cap structure was suggested in these RNAs. The cell-free protein synthesis using the archaebacterial extract was inhibited by anisomycin, a specific inhibitor for eukaryotic ribosomes. Two unique membrane-bound ATPases were detected. Based on resistance to H⁺-ATPase inhibitors, these enzymes seemed not to be F₀F₁-ATPase.     

Ribosome subunit to polysome ratios affect the synthesis of rRNA in Drosophila cells

Many investigations have revealed that ribosome numbers increase in parallel with the growth rate of cells. Here we show that the absolute level of protein synthesis may not be the only factor influencing rRNA synthesis in a nondividing eukaryotic cell. Under conditions of complete (greater than 99%) inhibition of protein synthesis by four different antibiotics, there is a corresponding inhibition of rRNA synthesis. At lower levels of inhibition of protein synthesis (70%), a different effect of individual antibiotics on rRNA synthesis is observed. Cycloheximide and anisomycin, which cause a decrease in the free subunit pool due to a buildup of polysomes, stimulate rRNA synthesis, whereas puromycin and pactamycin, which cause an increase in the free subunit pool, cause a decrease in rRNA synthesis. These effects on rRNA synthesis are not solely due to a low level of completed proteins. Pactamycin treatment allows completed proteins to be made yet lowers rRNA labeling, while anisomycin treatment does not show synthesis of complete proteins yet increases rRNA labeling. The result suggest that eukaryotic cells may regulate ribosome synthesis in response to the number of free versus translating (polysomal) ribosomes as do Escherichia coli cells.     

In vitro incorporation of tritium into native DNA

An exchange method is described for producing tritium-labeled native DNA in vitro with minimal physical damage to the DNA. Tritium-labeled calf thymus DNA prepared in this way has a specific activity of about 100 μCi/mmole of nucleotide (i.e., about 2 × 10⁸ dpm/mmole). Sedimentation velocity at neutral and alkaline pH indicate that the product has an average of two single strand breaks per duplex molecule of molecular weight 6 × 10⁶ daltons. The optical and thermal denaturation properties of the product are those of native DNA. The method should be particularly useful for labeling DNA from organisms that cannot be labeled conveniently in vivo.     

Synthesis and stability of [3H] 5—demethoxyubiquinone-9

Radioactive [³H]5-demethoxyubiquinone-9 (3-methyl-2-nonaprenyl-6-methoxy-1,4-benzo-quione), an intermediate in the biosynthesis of ubiquione-9 by selected microorganisms and by the rat, has been synthesized. 4-Methyl-3-nitrophenol was converted to the corresponding anisole with [³H]methyl iodide and the anisole was then reduced to the corresponding aniline. Oxidation of 6-methyl-3-methoxy [³H]aniline with chromic acid gave the corresponding 1,4-benzo-quinone which was reduced and alkylated with solanesol in the presence of boron trifluorideetherate. Oxidation with ferric chloride gave two isomers, 5-demethoxyubiquinone-9 and 6-methyl-2-nonaprenyl-3-methoxy-1,4-benzoquinone which were separated by thin layer chromatography. The [³H]methoxyl-5-demethoxyubiquinone-9 prepared had a specific radioactivity of 100 mCi/mmole.     

QUANTITATION OF [3H]THYMIDINE UPTAKE BY STIMULATED HUMAN LYMPHOCYTES†

We have investigated the relationship between cell numbers and the amount of tritiated thymidine ([³H]TdR) taken up by stimulated human peripheral lymphocytes, as a function both of labeling time and of the specific activity of the thymidine. Cells responding either to mitogens or to allogenic cells show simple first order kinetics for the uptake of thymidine. Fitting the data to a Michaelis-Menten type of model, we observe for labeling times of 12 hr and longer, non-competitive inhibition of thymidine uptake by increased specific activity of tritium label, regardless of the mode of stimulation. However, for an individual responder in MLC at any arbitrary but fixed specific activity, dose of [³H]TdR and labeling interval, we still observe a linear relationship between cell mass and incorporated label. In contrast, if specific responding combinations in mixed lymphocyte culture are compared, the inhibition by specific activity at longer time intervals becomes significant and influences the quantitative interpretation of results. Specific activities of less than 10 Ci/mmole and labeling times of 6 hr or less avoid inhibition and ensure a linear relationship between dividing cell number and CPM (counts per minute recorded) of incorporated label.     

Mutations in Methanobacterium formicicum conferring resistance to anti-80S ribosome-targeted antibiotics

Summary Mutants of Methanobacterium formicicum resistant to the anti-80S ribosome-targeted inhibitor anisomycin were isolated and characterized. The resistance phenotype is correlated with a mutationally altered 50S ribosomal subunit. Anisomycin resistance in the mutants is accompanied by cross-resistance to other inhibitors of the 80S peptidyl-transferase centre like narciclasine, bruceantin, trichodermin and verrucarin A and by hypersensitivity to sparsomycin. This phenotype is identical to that reported for anisomycin-resistant mutants of yeast; it appears therefore, that the anisomycin interaction sites on the 70S ribosomes from M. formicicum bear the structural features typical of eukaryotic 80S organelles.     

Puromycin photoaffinity labels small- and large-subunit proteins at the A site of the Drosophila ribosome

[3H]Puromycin covalently incorporates into the protein and to a much lesser extent into the RNA components of Drosophila ribosomes in the presence of 254-nm light. The photoincorporation reaction takes place with a small number of large- (L2 and L17) and small- (S8 and S22) subunit proteins as determined by two-dimensional gel analysis. More quantitative one-dimensional gel results show that puromycin reacts with each of these proteins in a functional site specific manner. The small percentage of the total labeling that occurs with rRNA also appears to be site specific. The rRNA labeling arises from a puromycin-mediated cross-linking of ribosomal protein and rRNA. Ionic conditions shift the pattern of puromycin-labeled ribosomal proteins. These results suggest that puromycin can occupy two distinct sites on Drosophila 80S ribosomes. The pattern of ribosomal proteins labeled by puromycin is affected by the presence of other antibiotics such as emetine, anisomycin, and trichodermin.     

The tritium labeling of small amounts of protein for analysis by electrophoresis on sodium dodecyl sulfate--polyacrylamide slab gels.

A simple and reproducible method for the tritium labeling of small amounts of proteins prior to analysis under denaturing conditions on polyacrylamide slab gels is described. The method involves the in vitro labeling of proteins by reductive methylation using formaldehyde and high specific activity [³H]potassium borohydride. Labeled proteins were detected by fluorography after fractionation on polyacrylamide slab gels in the presence of sodium dodecylsulfate. The overall procedure allows the analysis and molecular weight estimation of submicrogram quantities of protein.     

Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters

Nikkomycins and gougerotin are peptidyl nucleoside antibiotics with broad biological activities. The nikkomycin biosynthetic gene cluster comprises one pathway-specific regulatory gene (sanG) and 21 structural genes, whereas the gene cluster for gougerotin biosynthesis includes one putative regulatory gene, one major facilitator superfamily transporter gene, and 13 structural genes. In the present study, we introduced sanG driven by six different promoters into Streptomyces ansochromogenes TH322. Nikkomycin production was increased significantly with the highest increase in engineered strain harboring hrdB promoter-driven sanG. In the meantime, we replaced the native promoter of key structural genes in the gougerotin (gou) gene cluster with the hrdB promoters. The heterologous producer Streptomyces coelicolor M1146 harboring the modified gene cluster produced gougerotin up to 10-fold more than strains carrying the unmodified cluster. Therefore, genetic manipulations of genes involved in antibiotics biosynthesis with the constitutive hrdB promoter present a robust, easy-to-use system generally useful for the improvement of antibiotics production in Streptomyces.     

Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-³H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [³H]thymidine experiments and compared the degree of nonspecific labeling by [³H]adenine with that derived from [³H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.     

Stm1p alters the ribosome association of eukaryotic elongation factor 3 and affects translation elongation

Stm1p is a Saccharomyces cerevisiae protein that is primarily associated with cytosolic 80S ribosomes and polysomes. Several lines of evidence suggest that Stm1p plays a role in translation under nutrient stress conditions, although its mechanism of action is not yet known. In this study, we show that yeast lacking Stm1p (stm1Δ) are hypersensitive to the translation inhibitor anisomycin, which affects the peptidyl transferase reaction in translation elongation, but show little hypersensitivity to other translation inhibitors such as paromomycin and hygromycin B, which affect translation fidelity. Ribosomes isolated from stm1Δ yeast have intrinsically elevated levels of eukaryotic elongation factor 3 (eEF3) associated with them. Overexpression of eEF3 in cells lacking Stm1p results in a growth defect phenotype and increased anisomycin sensitivity. In addition, ribosomes with increased levels of Stm1p exhibit decreased association with eEF3. Taken together, our data indicate that Stm1p plays a complementary role to eEF3 in translation.     

Combined gene cluster engineering and precursor feeding to improve gougerotin production in Streptomyces graminearus

Gougerotin is a peptidyl nucleoside antibiotic produced by Streptomyces graminearus. It is a specific inhibitor of protein synthesis and exhibits a broad spectrum of biological activities. Generation of an overproducing strain is crucial for the scale-up production of gougerotin. In this study, the natural and engineered gougerotin gene clusters were reassembled into an integrative plasmid by λ-red-mediated recombination technology combined with classic cloning methods. The resulting plasmids pGOU and pGOUe were introduced into S. graminearus to obtain recombinant strains Sgr-GOU and Sgr-GOUe, respectively. Compared with the wild-type strain, Sgr-GOU led to a maximum 1.3-fold increase in gougerotin production, while Sgr-GOUe resulted in a maximum 2.1-fold increase in gougerotin production. To further increase the yield of gougerotin, the effect of different precursors on its production was investigated. All precursors, including cytosine, serine, and glycine, had stimulatory effect on gougerotin production. The maximum gougerotin yield was achieved with Sgr-GOUe in the presence of glycine, and it was approximately 2.5-fold higher than that of the wild-type strain. The strategies used in this study can be extended to other Streptomyces for improving production of industrial important antibiotics.