David Vazquez

Substrate and antibiotic binding sites at the peptidyl transferase centre of E. coli ribosomes

A number of antibiotic inhibitors of protein synthesis interfere with the function of the peptidyl transferase centre on the larger ribosomal subunit [ 1: review] . One of the mechanisms by which such inhibitors might act is competition for substratebinding at the A-site or the P-site on the peptidyl transferase centre. In order to investigate this possibility, and to analyse further the mechanism of the peptidyl transfer reaction, we have developed assays for substrate-binding. There is evidence that only the terminal moeities of tRNA interact with the peptidyl transferase centre (fig. l), and that other parts of the tRNA molecules perform other functions. In order to be specific, an assay for substrate-interaction at the centre should therefore employ substrates containing only the terminal portion of tRNA. The present paper reports an assay for substrate-interaction at the P-site, which is based on the measurement of binding of CACCA-Leu-Ac to 50 S subunits in the presence of ethanol. The interaction is blocked by certain antibiotic inhibitors of peptidyl transferase but not others. In conjunction with studies on binding at the A-site [6-81, results suggest that a number of antibiotics act at both the A-site and the P-site of peptidyl transferase while others act only at the A-site. A preliminary report of this work has been presented elsewhere [7].

Inhibitors of protein synthesis in eukaryotic cells: Comparative effects of some Amaryllidaceae alkaloids

The effects of eighteen compounds obtained from bulbs of the Amaryllidaceae family were tested on (a) animal cell growth, (b) DNA, RNA and protein synthesis by intact cells and (c) protein synthesis in cell-free systems. Dihydrolycorine, haemanthamine, lycorine, narciclasine, pretazettine and pseudolycorine halted HeLa cell growth at 10(-1) mM or lower concentrations. These compounds at their growth inhibitory concentrations block protein synthesis in ascites cells and stabilize HeLa cell polysomes in vivo. Endomyocarditis virus RNA-directed cell-free polypeptide synthesis by an ascites S-30 extract and acetyl-[14C]leucyl-puromycin formation by ascites ribosomes are also inhibited by the six compounds indicated above. It is therefore concluded that they halt protein synthesis in eukaryotic cells by inhibiting the peptide bone formation step.

Substrate and antibiotic binding sites at the peptidyl transferase centre of E. coli ribosomes: Binding of UACCA-Leu to 50 S subunits

In the past years we have been studying the peptidy1 transferase centre of the larger ribosomal subunit [l-3 review] . This centre is responsible for the catalysis of peptide bond formation in protein synthesis. The peptidyl transfer reaction presumably takes place in several steps, amongst which are the binding of the peptidyl donor and acceptor substrates at the Pand the A-site on the peptidyl transfer centre. There is ample evidence that only the terminal moieties of tRNA interact with the centre, and we have been developing a system to resolve the binding steps using simple substrates containing the CpCpA oligonucleotides of tRNA. We have already reported a system for study of substrate-interaction at the P-site of the peptidyl transferase centre, based on the measurement of CACCA-Leu-Ac binding to 50 S ribosomal subunits in the presence of ethanol. Using this system, substrate-binding at the P-site was shown to be inhibited by a number of antibiotic inhibitors of peptidy1 transfer but unaffected or even stimulated by others [4,5]. The present work deals with a complementary assay for substrate-binding at the A-site. The assay is similar to that for substrate-binding at the P-site but uses UACCA-Leu (or CACCA-Leu) instead of CACCA-Leu-Ac. The present results, together with those of another study [6,7], suggest that certain antibiotic inhibitors of peptidyl transfer act on both the Aand the P-site of the peptidyl transferase centre, others act only on one of the sites, and yet others act at neither site. A preliminary report of this work has already been published [8].

Simultaneous ribosomal resistance to trichodermin and anisomycin in Saccharomyces cerevisiae mutants.

A spontaneous mutant of Saccharomyces cerevisiae resistant to trichodermin has been isolated. It displays cross resistance both in vivo and in vitro to a number of sesquiterpene antibiotics (fusarenon X, trichothecin and verrucarin A) and to the chemically unrelated antibiotic anisomycin. The mutation conferring resistance to anisomycin and trichodermin is expressed in the 60-S subunit of the yeast 80-S ribosome. Mutant ribosomes bind [-14C]trichodermin much less efficiently than wild type ribosomes, suggesting that resistance may be due, at least in part, to this property. However, both types of ribosomes bind [-3H] anisomycin equally. These results suggest that anisomycin and trichodermin have different binding sites on the 60-S subunit of eukaryotic ribosomes, even though previous results have shown that both antibiotics bind to mutually exclusive sites.

Effects of some proteins that inactivate the eukaryotic ribosome

The glycoproteins abrin and ricin ([l] review) and the proteins PAP [2], alpha sarcin (J. E. Davies, personal communication), cretin II [3], curcin II [3] , enomycin [4] and phenomycin [S] have been reported to block translation by eukaryotic ribosomes. The glycoproteins abrin and ricin are very active in intact cells since they are composed of two subunits, one of which (the B chain) facilitates the entrance into the cell of the other subunit (the A chain), which catalytically inactivates the 60 S ribosomal subunits ([ 1 ] review). However ricin A chain and the individual proteins abrin A chain, PAP, alpha sarcin, cretin II, curcin II, enomycin and phenomycin are very active in blocking translation in cell-free systems but far less so in intact cells probably owing to the cellular permeability barrier [l--S]. All these toxins might have a similar or related mechanism of action since abrin A chain [6], ricin A chain [6], PAP [2], cretin 11 [3], alpha sarcin (J. E. Davies, personal communication) and enomycin [4,7] were observed to have a certain inhibitory effect on the EF l-dependent binding of aminoacyl-tRNA to the ribosome. However the mode of action of the toxins has been studied independently by different groups using different cells and cell-free systems and it is not possible to conclude from the results available whether or not the toxins act in a similar manner. We have therefore studied comparatively the effects of ricin and abrin with PAP, alpha sarcin and enomycin on the EF l-dependent binding of aminoacyl-tRNA, formation of the EF 2-GTP-ribosome complex, peptidyl-tRNA translocation and EF 2and ribosome-dependent GTP hydrolysis using

The trichodermin group of antibiotics, inhibitors of peptide bond formation by eukaryotic ribosomes

Abstract We have studied the effects of a number of sesquiterpene antibiotics of the trichodermin group on different steps of the elongation phase in human tonsils and yeast cell-free systems. We have observed that these antibiotics are more active in the human tonsils than in the yeast cell-free systems. When a similar system is considered the antibiotic trichodermin is more active than its derived alcohol trichodermol. Sesquiterpene antibiotics of the trichodermin group do not affect non-enzymatic binding of Ac-Phe-tRNA and either enzymatic or non-enzymatic binding of Phe-tRNA to human tonsil ribosomes. These antibiotics are also without effect on translocation to the P site of Ac-Phe-tRNA bound to the ribosomal A site. All the sesquiterpene antibiotics of the trichodermin group tested (trichodermin, trichodermol, verrucarin A, fusarenon X) are very active inhibitors in the peptide bond formation step. This inhibitory effect of antibiotics of the trichodermin group was observed in the “puromycin reaction” and in the “fragment reaction” assays. Binding of the substrates CACCA-Leu-Ac and UACCA-Leu, respectively, to the donor and the acceptor sites of the peptidyltransferase centre was also studied in this work. We have observed in this system an inhibitory effect in binding of substrates to both the donor and the acceptor site of the peptidyltransferase centre by all the antibiotics of the trichodermin group.

Bruceantin, a novel inhibitor of peptide bond formation.

Abstract The effects of bruceantin on a number of steps of the protein synthesis process have been studied using resolved model systems from both yeast and reticulocytes. Bruceantin is a potent inhibitor of polyphenylalanine synthesis as directed by poly(U). However, inhibition is less pronounced on protein synthesis as directed by endogenous mRNA and the compound inhibits the poly(U) system only poorly if added after polyphenylalanine synthesis has been initiated. Peptide bond formation as assayed in both the fragment reaction and in the puromycin reaction with a preformed initiation complex containing ribosomes and [ 35 S]Met-tRNA F is totally blocked by bruceantin. Neither the enzymic binding of Phe-tRNA to reticulocyte ribosomes nor the formation of the 35 S-labeled tRNA · ribosome initiation complex is inhibited by bruceantin. The binding of [ 14 C]trichodermin to yeast ribosomes is strongly inhibited by bruceantin. A Klotz plot shows that both these drugs bind to ribosomes in mutually exclusive fashion and it can be calculated that bruceantin binds to the peptidyltransferase center with K d = 0.34 μ M. This high affinity is considerably lower for polyribosomes ( K d = 557 μ M), which may explain the earlier finding that bruceantin only stabilizes polyribosomes at high drug concentrations.

Ribosome changes during translation

Abstract 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 m m -sodium azide treatment or 8 °C incubation of spheroplasts) or puromycin-dependent released ribosomes, have an affinity defined by Kd = 3.3 to 3.6 μ m . Ribosomes forming polysomes engaged in protein synthesis have at least two new different conformations (defined by Kd,H = 0.81 μ m and Kd,L = 12 μ m ). 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 KdCHX = 12 μ m and post-translocated polysomes (polysomes blocked with doxycycline) have an affinity of KdDC = 0.82 μ m . These dissociation constants are identical to Kd,L and Kd,H obtained with control untreated polysomes, respectively. Moreover, a new ribosome conformation defined by KdDT = 1.5 μ m and KdFA = 1.8 μ m 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.

Interaction between A 3-nitrobenzothiazolo (3,2-a) quinolinium antitumour drug and deoxyribonucleic acid

The interaction of 3-nitrobenzothiazolo (3,2-a) quinolinium (NBQ) perchlorate with DNA was studied by u.v.-visible and fluorescence spectrophotometry as well as by hydrodynamic methods. On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts, and the fluorescence was quenched. Binding parameters, determined from spectrophotometric measurements by Scatchard analysis according to an excluded-site model, indicated a binding constant of 2.4 X 10(5)M-1 for calf thymus DNA at ionic strength 0.01. The interaction was markedly suppressed by increasing the salt concentration. Binding to the GC-rich DNA of Micrococcus lysodeikticus was weaker than the binding to calf thymus DNA at ionic strength 0.01 NBQ increased the viscosity of sonicated rod-like DNA fragments, producing a calculated increment in length of 2.4 A/bound drug molecule. It removed and reversed the supercoiling of closed circular duplex plasmid pBR322 DNA by virtue of a helix-unwinding angle estimated as approximately 13 degrees/bound ligand molecule. We conclude that the binding of NBQ to DNA occurs by a mechanism of intercalation, which probably accounts for its reported antitumor activity.

Location of resistance to the alkaloid narciclasine in the 60S ribosomal subunit

Narciclasme is an alkaloid of known chemical structure and antitumour activity obtained from bulbs of different species of Narcissus [1-3]. In preliminary studies we have observed that narciclasine inhibits growth of cells of Ehrlich ascites tumour by immediately halting protein synthesis (unpublished results). This effect results from specific inhibition of the step of peptide bond formation [4], The results presented in this paper show that resistance to narciclasine in a mutant strain of Saccharomyces cerevisiae is due to an alteration on the peptidyl transferase centre of the 60S ribosomal subunit.