David Vázquez

Inhibition of ribosomal translocation by aminoglycoside antibiotics

Abstract The translocation of AcPhe-tRNA in a purified system and that of peptidyl-tRNA in a crude, complete polypeptide synthesizing system containing endogenous E. coli polysomes are inhibited by antibiotics of the neomycin, kanamycin and gentamicin groups. The extent of inhibition varies with the different antibiotics, but it correlates well with the capacity of each antibiotic to inhibit polypeptide chain elongation. Thus, the inhibition of translocation by these antibiotics is clearly significant for their inhibitory effect on polypeptide synthesis.

The mode of action of the antitumor drug bouvardin, an inhibitor of protein synthesis in eukaryotic cells.

Bouvardain is an antitumor drug that inhibits protein synthesis in intact eukaryotic cells and cell‐free systems. Our present studies have shown that bouvardin acts at the level of the 80 S ribosome in a site somehow involved with the interaction of EF1 and EF2. Indeed bouvardin inhibits EF1‐dependent binding of aminoacyl‐tRNA and EF2‐dependent translocation of peptidyl‐tRNA but does not affect the non‐enzymic translocation since this relation does not require EF2. The site of the 80 S ribosome involved in the interaction with bouvardin appears to be independent from the cycloheximide and the cryptopleurine binding sites since yeast mutants resistant to cycloheximide or cryptopleurine are sensitive to bouvardin.

Intercalative binding to DNA of antitumour drugs derived from 3-nitro-1,8-naphthalic acid.

Two new antitumour drugs, imide derivatives of 3-nitro-1,8-naphthalic acid having different basic side chains linked to the imide nitrogen, have been shown to bind to double-helical DNA by intercalation. At ionic strength 0.01 mol/litre, pH 7, their intrinsic association constants are about 1.45 x 10(5) M-1 and each bound ligand molecule occludes about 3.4 nucleotides of the DNA lattice. They remove and reverse the supercoiling of closed circular duplex PM2 DNA with apparent unwinding angles of 11-12 degrees per bound drug molecule, referred to an assumed unwinding angle of 26 degrees for ethidium. They increase the viscosity of sonicated rod-like DNA fragments, each bound drug molecule producing a calculated increment in length of 2.2 - 2.5 A. No important differences between the DNA-binding characteristics of the two drugs were detected, though one appears marginally more active than the other in certain biological tests.

Narciclasine: an antitumour alkaloid which blocks peptide bond formation by eukaryotic ribosomes

The antitumour activity of crude preparations of bulbs from species of Narcissus [l] is due to the alkaloid narciclasine, of known chemical structure [2,3], which exerts an antimitotic effect during metaphase [4]. Studies in our laboratory have shown that narciclasine immediately halts protein synthesis in Ehrlich ascites tumour and Saccharomyces cerevisiae cells without affecting RNA synthesis (A. Jimenez, personal communication). The results presented in this communication show that narciclasine inhibits protein synthesis in rabbit reticulocyte and yeast cell-free systems by blocking peptide bond formation at the ribosome level.

Activities of ribosomal cores deprived of proteins L7, L10, L11 and L12

The protein Ll 1 of the large ribosomal subunit from Escherichia coli has been reported to be involved in the peptidyl transferase activity of the ribosome [ 11. Thus protein Ll 1 was postulated as either a part of the active center or even as the actual peptidyl transferase itself [2]. Recently Highland and Howard [3] have described the preparation of 50s subunit derived core particles deprived of proteins L7, Ll 0, Ll 1 and L12 following basically the method of Hamel et al. [4]. Because of the low number of proteins separated in the treatment, these particles are very useful for studies on the role of the split proteins on the ribosomal activities. We have investigated the role of protein Ll 1 on the activities of the peptidyl transferase centre using the Ll l-deprived cores and the results are reported in this communication.

Reconstitution of the 50S ribosome subunit. Role of proteins L 7 and L 12 in the GTPase activities. Site of action of thiostrepton

Upon treatment of Escherichia coli ribosomes with 1 M ammonium chloride in the presence of 50% ethanol, ribosome-derived protein deficient particles are obtained. These particles are inactive for the GTPase activities dependent on ribosomes and either elongation factor G (EF G) or elongation factor T (EF T) [l] _ The deficient particles recover these GTPase activities by addition of the released proteins [l-4] which have a strong acidic character and have been identified as proteins L 7 and L 12 [3, 41 following the nomenclature of Wittmann [5] . The same two proteins have been independently isolated and studied by Mijller and coworkers [6,7] ; proteins L 7 and L 12 were also found in their systems to be involved in the recovery of the G-dependent GTP hydrolytic activity in the reconstitution of 50 S-derived cores deprived of a number of proteins [6]. We have studied the effect of alcohols in EF Gand EF Tdependent functions of the 50 S ribosome subunit. Ethanol and methanol strongly stimulate the EF G-dependent hydrolysis of GTP in the presence of 50 S-derived cores obtained by dissociation of some proteins in CsCl gradients [8] . The alcohol effect is more relevant in studies on EF T-dependent activities. EF T-dependent binding of Phe-tRNAphe to ribosomes is hardly affected by moderate concentrations of methanol whereas the GTP hydrolysis is considerably enhanced under the same conditions. This effect results in an uncoupling of the EF T-dependent aminoacyltRNA binding and the GTP hydrolysis [9] which are believed to take place normally in the proportion 1: 1. Furthermore this methanoland EF T-dependent

Functional interaction of neomycin B and related antibiotics with 30S and 50S ribosomal subunits.

Abstract Antibiotics of the neomycin, kanamycin and gentamicin, but not streptomycin, groups stabilize the GDP·elongation factor (EF) G·50S subunit·fusidic acid complex. Treatment of 30S subunits, but not of 50S subunits, with neomycin B or kanamycin B, followed by removal of excess unbound antibiotic and supplementation with untreated complementary subunits, promotes poly(U)-dependent binding of Tyr-tRNA to the reassociated ribosomes (misreading). A similar treatment of either ribosomal subunit with neomycin B inhibits the EF-G-dependent translocation of Ac-Phe-tRNA. These results suggest that interaction of neomycin B and related antibiotics with the 30S subunit induces misreading and inhibits translocation, and interaction with the 50S subunit stabilizes EF-G on the ribosome and also inhibits translocation.

Acidic proteins from Saccharomycescerevisiae ribosomes

Abstract Two dimensional gel electrophoresis of ribosomal proteins from Saccharomyces cerevisiae reveals the presence of three spots in the region corresponding to proteins of high acidic character. Washing the ribosomes with 0.4 M NH4Cl and 50% ethanol, followed by chromatography of the extracted proteins on DEAE-cellulose, indicated the presence of two fractions of acidic proteins; (A and Ax), having very similar molecular weights (12.000–13.000), but phosphorylated to different extents. Fractions A and Ax are immunologically distinct and their immunologic properties differ from acidic proteins found in Escherichia coli , rat liver, and Artemia salina ribosomes. Protein A can be resolved into two bands by electrofocusing, and two dimensional gel electrophoresis. The two components correspond to proteins L44 and L45 according to the standard nomenclature. Proteins Ax seems to correspond to the spot that moves above and to the left of L44 and L45 and is at least three times more phosphorylated than these two proteins.

Proteins associated with rRNA in the Escherichia coli ribosome

Ribosomal proteins located near the rRNA have been identified by cross linking to [14C]spermine with 1,5-difluoro-2,4-dinitrobenzene. The polyamine binds to double-stranded rRNA; those proteins showing radioactivity covalently bound after treatment with the bifunctional reagent should therefore be located in the vicinity of these regions of rRNA. Six proteins from the small subunit, S4, S5, S9, S18, S19 and S20 and ten proteins from the large subunit L2, L6, L13, L14, L16, L17, L18, L19, L22 and L27 preferentially take up the label. The results obtained with three proteins from the large subunit, L6, L16 and L27, show a high degree of variability that could reflect differences of conformation in the subunit population. Several proteins were drastically modified by the cross-linking agent but were not detected in the two-dimensional gel electrophoresis (e.g., S1, S11, S21, L7, L8 and L12) and therefore could not be studied.

Induction of cell lysis in Escherichia coli: cooperative effect of nocardicin A and mecillinam.

Nocardicin A and mecillinam are two beta-lactam antibiotics with poor bacteriolytic activity against Escherichia coli. However, the combined use of these drugs resulted in the induction of a fast lytic response in E. coli cells. For this cooperative effect to take place, the formation of a complex between penicillin-binding protein 2 and mecillinam is apparently necessary. This suggests that penicillin-binding protein 2 might be actively involved in the response of E. coli to bacteriolytic beta-lactam antibiotics.