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PubMed1977

Peptidyl transferase activity in wheat germ ribosomes. Effect of some antibiotics.

The formation of N-acetyl-leucyl-puromycin in a "fragment reaction" catalyzed by 80 S ribosomes from wheat germ was characterized. The reaction product was identified by high-voltage electrophoresis. The fragment reaction is inhibited by sparsomycin, blasticidin S, gougerotin and to a lesser degree by amicetin and tetracycline. Formation of an acLeu-pentanucleotide-ribosomes complex was strongly stimulated by sparsomycin.

Acyltransferases↗

PubMed2003

Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit.

Structures of anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bound to the large ribosomal subunit of Haloarcula marismortui have been determined at 3.0A resolution. Most of these antibiotics bind to sites that overlap those of either peptidyl-tRNA or aminoacyl-tRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H.marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA.

Anisomycin↗

PubMed1993

Hybrids of antibiotics inhibiting protein synthesis. Synthesis and biological activity.

Four hybrid antibiotics combining structural features of chloramphenicol (1a), sparsomycin (2b), lincomycin (5c), and puromycin (6d)--lincophenicol (1c), chloramlincomycin (5a), sparsolincomycin (5b), and sparsopuromycin (6b)--were synthesized. They were investigated as inhibitors of several partial reactions of procaryotic and eucaryotic protein synthesis as well as potential antimicrobial agents. Lincophenicol (1c) was active as inhibitor of Escherichia coli ribosomal peptidyltransferase-catalyzed puromycin reaction. Both lincophenicol (1c) and sparsophenicol (1b) inhibited the binding of the iodophenol analogue of sparsomycin to E. coli ribosomes. The results are discussed in terms of a retro-inverso hypothesis advanced earlier for interpretation of biological activity of chloramphenicol (1a) and sparsophenicol (1b). Chloramlincomycin (5a) suppressed the growth of Streptococcus pyogenes with MIC 6.25 micrograms/mL.

Animals↗

PubMed1991

Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state.

Base Sequence↗

PubMed2003

Catalytic properties of mutant 23 S ribosomes resistant to oxazolidinones.

Kinetic analysis of ribosomal peptidyltransferase activity in a methanolic puromycin reaction with wild type and drug-resistant 23 S RNA mutants was used to probe the structural basis of catalysis and mechanism of resistance to antibiotics. 23 S RNA mutants G2032A and G2447A are resistant to oxazolidinones both in vitro and in vivo with the latter displaying a 5-fold increase in the value of Km for initiator tRNA and a 100-fold decrease in Vmax in puromycin reaction. Comparison of the Ki values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of the peptidyltransferase A-site. Inhibition of the mutants using a truncated CCA-Phe-X-Biotin fragment as a P-site substrate is similar to that observed with the intact initiator tRNA, indicating that the inhibition is substrate-independent and that the peptidyltransferase itself is the oxazolidinone target. Mapping of all known mutations that confer resistance to these drugs onto the spatial structure of the 50 S ribosomal subunit allows for docking of an oxazolidinone into a proposed binding pocket. The model suggests that oxazolidinones bind between the P- and A-loops, partially overlapping with the peptidyltransferase P-site. Thus, kinetic, mutagenesis, and structural data suggest that oxazolidinones interfere with initiator fMet-tRNA binding to the P-site of the ribosomal peptidyltransferase center.

Antibiotics, Antineoplastic↗

PubMed1975

Inhibitors of protein synthesis V. Irreversible interaction of antibiotics with an initiation complex.

The initiation complex (t-complex) formed in a cell-free system (E. coli) from Ac-Phe-tRNA, poly(U) and washed ribosomes in the presence of initiation factors (ribosomal wash) and GTP, contains the Ac-Phe-tRNA bound quantitatively in a puromycin-reactive state. The t-complex is irreversibly inactivated by spiramycin with respect to its reactivity toward puromycin. The inactivated t-complex retains all of the Ac-Phe-tRNA bound, but it does not react with puromycin (2 x10-minus-3M) within 32 min at 25 degrees. In the case of another inhibitor protein synthesis, sparsomycin, the permanently "modified" t-complex not only retains all the bound Ac-Phe-tRNA but it can still react with puromycin. In the continuous presence of sparsomycin (1 x 10-minus-7M) the bound Ac-Phe-tRNA reacts quantitatively at a rate which is one-tenth the rate at which the t-complex reacts with puromycin, at low (6.25 x 10-minus-5M) or high (2 x 10-minus-3M) concentrations. These results are not in agreement with current views according to which aparsomycin binds to the ribosome reversibly at a single site with a KI in the range of 10-minus6-10-minus-7 M and according to which this stie is at the A'-site (puromycin site) of peptidyl transferase.

Antibiotics, Antineoplastic↗

PubMed1976

The binding of tritiated elongation-factors 1 and 2 to ribosomes from Krebs II mouse ascites-tumore cells. The influence of various antibiotics and toxins.

The effect of a number of different antibiotics and toxins on the capacity of Krebs II mouse ascites ribosomes to bind 3H-labelled elongation factors (EF-1 and EF-2) has been examined. It was found that abrin and ricin inhibit the binding of EF-2, while diphtheria toxin, sparsomycin, streptovitacin A, and cycloheximide had essentially no effect on its binding. Of the other compounds examined, sparsomycin was unique in its capacity, under some circumstances, to significantly affect the binding of aminoacyl-tRNA and EF-1 to ribosomes. Fusidic acid appears to nonspecifically enhance the binding of both EF-1 and EF-2.

Abrin↗

PubMed1979

Peptidyl transferase of bacterial ribosome: resistance to proteinase K.

70-S ribosomes and 50-S ribosomal subunits from Escherichia coli D10 were treated with proteinase K for increasing periods of time. Peptidyl transferase activity and sparsomycin-induced binding of (U)C-A-C-C-A-[3H]Leu-Ac were tested in the treated particles, the binding of the substrate being more sensitive to the protease than peptide bond formation. Comparison of the amounts of proteins present in the treated particles with the residual activity indicates that only proteins L3 and L14 are released at a similar rate to that at which peptidyl transferase activity is lost. Proteins related to this ribosomal activity by other techniques are lost at a faster rate than the activity itself. In addition, the results indicate that sparsomycin stimulates the binding of the substrate by a different mechanism from that which inhibits peptide bond formation.

Acyltransferases↗

PubMed1992

Use of 50 S-binding antibiotics to characterize the ribosomal site to which peptidyl-tRNA is bound.

Five antibiotics (puromycin, erythromycin, lincomycin, sparsomycin, and virginiamycin M1) that bind specifically to the 50 S ribosomal subunit near the peptidyl transferase center were used to compare and characterize the positions of bound AcylPhe-tRNA in the puromycin-reactive and -unreactive states. Binding of the antibiotics was quantitatively measured by their perturbation of fluorescence from probes attached to the alpha-amino group of Phe-tRNA. Derivatives of three probes with differing chemical characteristics and environmental sensitivities were used: a coumarin, an aminonaphthalenesulfonate, and a pyrene. The effects of the antibiotics on the fluorescence of labeled AcylPhe-tRNAs in the two states, while generally qualitatively similar, are nonetheless quantitatively distinct, as are the calculated binding constants for the antibiotics. Puromycin, as reported earlier, binds to both the puromycin-reactive and -unreactive states, but its dissociation constant is higher for the latter state. Erythromycin binds tightly to ribosomes bearing labeled AcylPhe-tRNA in either the puromycin-reactive or -unreactive state. Its effect on the fluorescence of the labeled tRNA is very similar in the two states, except with the pyrene probe, where it has a larger effect in the puromycin-reactive state. Lincomycin and sparsomycin bind to both ribosomal states, but both bind more tightly to the puromycin-reactive state, the extent of the difference varying with the identity of the fluorescent probe. Virginiamycin M1 binds to ribosomes with AcylPhe-tRNA in the puromycin-reactive site, but its binding could not be detected to ribosomes with AcylPhe-tRNA in the puromycin-unreactive site.

Anti-Bacterial Agents↗

PubMed2005

Ribosomal proteins cross-linked to the initiator AUG codon of a mRNA in the translation initiation complex by UV-irradiation.

Eukaryotic ribosomal proteins constituting the binding site for the initiator codon AUG on the ribosome at the translation initiation step were investigated by UV-induced cross-linking between protein and mRNA. The 80S-initiation complex was formed in a rabbit reticulocyte cell-free system in the presence of sparsomycin with radiolabeled Omega-fragment as a template, which was a 73-base 5'-leader sequence of tobacco mosaic virus RNA having AUG at the extreme 3'-terminal end and extended with 32pCp. Two radioactive peaks were sedimented by sucrose gradient centrifugation, one being the 80S initiation complex formed at the 3'-terminal AUG codon, and the other presumably a "disome" with an additional 80S ribosome bound at an upstream AUU codon, formed when Omega-fragment was incubated with sparsomycin [Filipowicz and Henni (1979) Proc. Natl. Acad. Sci. USA 76, 3111-3115]. Cross-links between ribosomal proteins and the radiolabeled Omega-fragment were induced in situ by UV-irradiation at 254 nm. After extensive nuclease digestion of the complexes, ribosomal proteins were separated by two-dimensional gel electrophoresis. Autoradiography identified the proteins S7, S10, S25, S29, and L5 of the 80S initiation complex and S7, S25, S29 and L5 of that in the disome as 32P-labeled proteins. Together with the results of cross-linking experiments of other investigators and recently solved crystal structures of prokaryotic ribosomes, the spatial arrangement of eukaryotic ribosomal proteins at the AUG-binding domain is discussed.

Animals↗

PubMed1979

Effect of inhibitory and stimulatory agents on protein synthesis in hepatomas and host livers of rats.

Female inbred BUF rats bearing intrahepatically transplanted hepatomas (5123 or 19) were subjected to acute exposure to a variety of hepatotoxic agents (actinomycin D, aflatoxin B1, CCl4, dimethylnitrosamine, ethionine, puromycin, or sparsomycin) or of stimulatory agents (hydrocortisone, phenobarbital, or whole-body X-ray). The responses in terms of changes in polyribosomes and protein synthesis (in vitro and in vivo) of host liver and hepatoma were evaluated. The responses of the host livers and hepatomas to the different agents varied. In general, the host livers responded much more than did the hepatomas. Of the two hepatomas, hepatoma 19 responded less (particularly in terms of polyribosome changes) than did hepatoma 5123. In a few experiments, different doses of actinomycin D, ethionine, or sparsomycin were used and in all instances the host livers responded more than did the hepatomas.

Animals↗

PubMed1977

The effect of translation and transcription inhibitors on the synthesis of periplasmic phosphatases in E. coli.

Previous studies by others have indicated that the synthesis of secreted enzymes is unusually sensitive to many translation inhibitors and resistant, for about 30 min, to rifampicin. We have studied the sensitivity of secreted (periplasmic) phosphatases to such inhibitors. Alkaline phosphatase synthesis is more sensitive than total protein synthesis to tetracyclin and spectinomycin, but not to sparsomycin, streptomycin, chloramphenicol, kasugamycin, blasticidin S or thiostrepton; it is slightly more resistant than total protein synthesis to the latter two antibiotics. Acid hexose-phosphatase was also preferentially sensitive to tetracyclin and spectinomycin and also to kasugamycin. beta-galactosidase was also included in the study, as an intracellular enzyme, and was found to be preferentially inhibited ("repressed"), sometimes transiently, by all eight translation inhibitors. This effect did not seem to be mediated through cyclic AMP or guanosine tetraphosphate; the "repression" was still evident in mutants with altered rho factor indicating that it may also not be related to artificial polarity. Synthesis of both periplasmic phosphatases was immediately inhibited by rifampicin. These results differ from those found in previous studies with other organisms and suggest a reappraisal of the usual interpretation of these phenomena.

Acid Phosphatase↗

PubMed1993

Correlation of the in vitro cytotoxicity of ethyldeshydroxysparsomycin and cisplatin with the in vivo antitumour activity in murine L1210 leukaemia and two resistant L1210 subclones.

The cultured murine leukaemia L1210 cell populations used in the present study were derived from L1210 cells that had been grown in vivo. Subclones resistant to sparsomycin (L1210/Sm) or cisplatin (L1210/CDDP) were also developed in vivo. The doubling times of the cultured cell populations were identical. Fractions surviving after drug treatment in vitro were determined by colony formation in soft agar. The results, based on the differential sensitivity of the cell populations to ethyldeshydroxysparsomycin (EdSm) and CDDP, indicated that after a short exposure, cultured L1210/CDDP cells were cross-resistant to EdSm. L1210/Sm cells, however, were not cross-resistant to CDDP. The results obtained in cultured cell populations were confirmed in vivo. CD2f1 mice bearing i.p. implants of 1 x 10(5) tumour cells were given EdSm or CDDP and a combination of the two agents. Drugs were given once daily every 4 days for 3 doses starting at 24 h after tumour implantation. Treatment of mice bearing L1210/wt leukaemia with combined EdSm and CDDP caused strongly synergistic antitumour activity. In animals bearing the two resistant subclones, however, combined drug treatment did not improve the antitumour activity. The corresponding median survival of mice receiving combined drug treatment was 60 days in each group containing 6 mice bearing L1210/wt, with 4-6 cures being noted; 19 days in animals harbouring L1210/Sm, with 2 cures being recorded among 6 mice; and 11 days in mice bearing L1210/CDDP, with no cure being obtained. The results of this study indicate that the synergism resulting from combined treatment with CDDP and EdSm is a function of the cellular properties of the target tumour-cell populations and is independent of host factors.

Animals↗

PubMed1984

Superinduction of human interleukin-2 messenger RNA by inhibitors of translation.

Stimulation of human lymphocytes with phytohemagglutinin results in the induction of messenger RNA encoding interleukin-2 (IL-2), a lymphokine possessing immuno-regulatory properties. We have previously demonstrated a dramatic superinduction of the formation of IL-2 and of biologically active IL-2 mRNA in the presence of cycloheximide, an inhibitor of translation. These findings suggested the involvement of a labile protein repressor in the regulation of human IL-2 gene expression. Here, we show a commensurate superinduction of IL-2 mRNA sequences by three additional inhibitors of protein synthesis with distinct modes of action: T-2 toxin, pactamycin, and sparsomycin. These results strengthen the concept that a labile protein controls the formation of mature IL-2 mRNA. They tend to eliminate the possibility that the superinduction phenomenon observed in the presence of cycloheximide is due to its action on a process other than translation.

Cells, Cultured↗

PubMed1978

The effect of inhibitors of protein synthesis on the reexpression of surface immunoglobulin following antigenic modulation.

P3, a cell line derived from the plasmacytoma MOPC-21 secretes IgG1 (K) and is sensitive to complement (C')-mediated lysis by antibody directed against gamma1 or K. Sensitivity is attributed to the presence of immunoglobulin molecules on the surface membrane, designed Ig-mem. This sensitivity is abolished by antigenic modulation of Ig-mem. Modulated cells, when incubated in the absence of antibody, recover sensitivity to lysis in 4 hr. By measuring the rate of recovery, it has been possible to study the effects of various drugs on the reexpression of Ig-mem. Treatment of modulated cells with cycloheximide (Cx), pactamycin Pc), anisomycin (An), homoharringtonine (Ha) or sparsomycin (Sm), each a specific inhibitor of a different step in protein synthesis, produces a significant reduction in the rate of recovery. Paradoxically, puromycin (Pm), also a specific inhibitor of protein synthesis, does not reduce the rate of recovery. Studies were performed using Pm together with each of the other drugs to gain an understanding of the relationship between protein synthesis and recovery from modulation. Based upon these studies, we conclude that continued operation of the initiation cycle of protein synthesis is required for reexpression of Ig-mem in the absence of de novo protein formation.

Anisomycin↗

PubMed2003

Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression.

Crystal structures of tRNA mimics complexed with the large ribosomal subunit of Deinococcus radiodurans indicate that remote interactions determine the precise orientation of tRNA in the peptidyl-transferase center (PTC). The PTC tolerates various orientations of puromycin derivatives and its flexibility allows the conformational rearrangements required for peptide-bond formation. Sparsomycin binds to A2602 and alters the PTC conformation. H69, the intersubunit-bridge connecting the PTC and decoding site, may also participate in tRNA placement and translocation. A spiral rotation of the 3' end of the A-site tRNA around a 2-fold axis of symmetry identified within the PTC suggests a unified ribosomal machinery for peptide-bond formation, A-to-P-site translocation, and entrance of nascent proteins into the exit tunnel. Similar 2-fold related regions, detected in all known structures of large ribosomal subunits, indicate the universality of this mechanism.

Bacterial Proteins↗

PubMed1977

Mutants of CHO cells resistant to the protein synthesis inhibitors, cryptopleurine and tylocrebrine: genetic and biochemical evidence for common site of action of emetine, cryptopleurine, tylocrebine, and tubulosine.

Stable mutants resistant to the protein synthesis inhibitors cryptopleurine and tylocrebine can be isolated in Chinese hamster ovary (CHO) cells, in a single step. The frequency of occurrence of cryptopleurine (CryR) and tylocrebrine (TylR) resistant mutants in normal and mutagenized cell populations is similar to that observed for emetine resistant (EmtR) mutants. The CryR, TylR, and EmtR mutants exhibit strikingly similar cross-resistance to the three drugs used for selection, to tubulosine and also to two emetine derivatives cephaeline and dehydroemetine, based on assays of in vivo cytotoxicity and on assays of protein synthesis in cell-free extracts. The identity of cross-resistance patterns of the CryR, TylR, and EmtR mutants indicates that the resistance to all these compounds results from the same primary lesion, which in the case of EmtR cells has been shown to affect the 40S ribosomal subunit. This conclusion is strongly supported by the failure of EmtR, TylR, and CryR mutants to complement each other in somatic cell hybrids. Based on these results it is suggested that the above group of compounds possesses common structural determinants which are responsible for their activity. The above mutants, however, do not show any cross-resistance to other inhibitors of protein synthesis such as cycloheximide, trichodermin, anisomycin, pactamycin, and sparsomycin, either in vivo or in vitro, indicating that the site of action of these inhibitors is different from that of the emetine-like compounds.

Alkaloids↗

PubMed1976

Analysis of the two steps in polypeptide chain initiation inhibited by pactamycin.

Earlier work has shown that the inhibition by pactamycin (PM) of polypeptide chain initiation in reticulocyte extracts is associated with (1) a defect in the joining of the 60S subunit to the smaller initiation complex to form an 80S complex ("joining reaction") (Kappen, L. S., Suzuki, H., and Goldberg, I. H. (1973), Proc. Natl. Acad. Sci. U.S.A. 70, 22) and (2) a block after the synthesis of the initial dipeptide (Kappen, L. S., and Goldberg, I. H. (1973), Biochem. Biophys. Res. Commun. 54, 1083). The relative contributions of these two effects to the action of PM and their relationship to one another were evaluated in a system employing sparsomycin that permits both initiation at a certain number of initiation sites and limited oligopeptide formation without termination and release. The degree to which PM blocks the "joining reaction" and leads to the accumulation of 48S initiation complexes that either remain free or are bound to polysomes without the corresponding 60S subunit ("half-mers") was estimated by treatment of polysomes with RNase. Met-tRNAfMet binding factors are required to stabilize the RNase-generated 48S complexes. Under conditions where the initiation factor required for the "joining reaction" functions catalytically, presumably by cycling on and off initiation complexes, PM usually inhibits 80S complex formation 50-70%. Where "joining" is not limiting (presence of at least stoichiometric amounts of joining factor or high Mg2+ concentration) PM leads to the maximal accumulation of the initial dipeptide, Met-Val, in the P-site on the ribosome, indicating a block in a subsequent step in elongation. Binding studies with [3H]PM and the inability of PM to inhibit elongation of preformed Met-Val indicate that PM must interact with the ribosomes at an early stage of initiation. Taken together these data are compatible with the suggestion that PM does not interfere with the ribosomal "joining reaction" per se, but prevents the release and reuse of the joining factor, and in so doing blocks a step in elongation after formation of the initial dipeptide and its translocation to the P-site on the ribosome.

Animals↗