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At least 19 recordsLinked to original sources
PubMed1996

Characterization of the covalent binding of thiostrepton to a thiostrepton-induced protein from Streptomyces lividans.

Thiostrepton is a highly modified multicyclic peptide antibiotic synthesized by diverse bacteria. Although best known as an inhibitor of protein synthesis, thiostrepton is also a potent activator of gene expression in Streptomyces lividans. In these studies, we characterize the nature of the interaction between thiostrepton and two proteins that it induces, TipAL and TipAS. In the absence of added cofactors, thiostrepton formed a complex with either TipAL or TipAS in aqueous solution. The TipA-thiostrepton complex was not dissociated by denaturants such as SDS, urea, or disulfide reducing agents. The mass of the TipAS-thiostrepton complex as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) was equivalent to the sum of TipAS and thiostrepton. Thiostrepton also reacted spontaneously with free cysteine (but not with other amino acids tested) to generate stable compounds having masses equivalent to thiostrepton plus 3 to 4 cysteines. Blocking experiments indicated that complex formation required dehydroalanine residues on thiostrepton and cysteine residues on TipAS. When the TipAS-thiostrepton complex was digested with trypsin and analyzed by MS, the thiostrepton adduct was found bound only to the unique cysteine-containing TipAS peptide fragment. Amino acid analysis confirmed that the TipAS-thiostrepton complex contained lanthionine, the product of a reaction between dehydroalanine and cysteine. Together, these data document a covalent attachment of thiostrepton to TipA proteins mediated by bond formation between dehydroalanine of thiostrepton and cysteine of TipAS. Implications regarding the function of TipAS as a thiostrepton (electrophile)-sequestering protein and thiostrepton-mediated activation of TipAL as a model of irreversible transcriptional activation are discussed.

Alanine↗
PubMed1976

Binding of thiostrepton to ribosomes from thiostrepton-sensitive and -resistant Bacillus subtilis strains.

Binding of [(35)S]thiostrepton to ribosomes from thiostrepton-sensitive and -resistant strains of Bacillus subtilis was studied. Ribosomes from thiostrepton-resistant strains bound relatively little thiostrepton compared with ribosomes from thiostrepton-sensitive B. subtilis. In addition, ribosomes from revertant strains that were obtained as thiostrepton-sensitive revertants from thiostrepton-resistant strains bound [(35)S]thiostrepton similarly to ribosomes from the sensitive parental strain.

Anti-Bacterial Agents↗
PubMed1976

Thiostrepton-resistant mutants of Bacillus subtilis: localization of resistance to the 50S subunit.

A number of thiostrepton-resistant mutants of Bacillus subtilis were obtained. The thi mutations map proximally to strA. Effects of thiostrepton on polyphenylalanine synthesis with ribosomes of S-100 fractions from parent and mutant strains indicated that resistance was localized to the ribosomes. Furthermore, effects of thiostrepton on binding of [3H]GTP to ribosomes and 50S subunits from thiostrepton-sensitive and -resistant strains localized the site of resistance to the 50S subunit. In addition, revertants from thiostrepton-resistance to thiostrepton-sensitivity were obtained. Ribosomes and 50S subunits from these thiostrepton-sensitive revertants were sensitive to thiostrepton similar to parental sensitive B. subtilis.

Anti-Bacterial Agents↗
PubMed1994

The antibiotics micrococcin and thiostrepton interact directly with 23S rRNA nucleotides 1067A and 1095A.

The antibiotics thiostrepton and micrococcin bind to the GTPase region in domain II of 23S rRNA, and inhibit ribosomal A-site associated reactions. When bound to the ribosome, these antibiotics alter the accessibility of nucleotides 1067A and 1095A towards chemical reagents. Plasmid-coded Escherichia coli 23S rRNAs with single mutations at positions 1067 or 1095 were expressed in vivo. Mutant ribosomes are functional in protein synthesis, although those with transversion mutations function less effectively. Antibiotics were bound under conditions where wild-type and mutant ribosomes compete in the same reaction for drug molecules; binding was analysed by allele-specific footprinting. Transversion mutations at 1067 reduce thiostrepton binding more than 1000-fold. The 1067G substitution gives a more modest decrease in thiostrepton binding. The changes at 1095 slightly, but significantly, lower the affinity of ribosomes for thiostrepton, again with the G mutation having the smallest effect. Micrococcin binding to ribosomes is reduced to a far greater extent than thiostrepton by all the 1067 and 1095 mutations. Extrapolating these results to growing cells, mutation of nucleotide 1067A confers resistance towards micrococcin and thiostrepton, while substitutions at 1095A confer micrococcin resistance, and increase tolerance towards thiostrepton. These data support an rRNA tertiary structure model in which 1067A and 1095A lie in close proximity, and are key components in the drug binding site. None of the mutations alters either the higher order rRNA structure or the binding of r-proteins. We therefore conclude that thiostrepton and micrococcin interact directly with 1067A and 1095A.

Anti-Bacterial Agents↗
PubMed1979

Ribosomal protein alterations in thiostrepton- and Micrococcin-resistant mutants of Bacillus subtilis.

Ribosomal proteins of parental thiostrepton- and micrococcin-sensitive Bacillus subtilis cysA14 and thiostrepton-and micrococcin-resistant mutants were compared. Several electrophoretic and immunochemical techniques showed unambiguously that BS-L11 was not present on 50 S ribosomal subunits from the six thiostrepton-resistant mutants. Protein BS-L11 reappeared in all six revertants from thiostrepton resistance to thiostrepton sensitivity. No definitive protein alteration could be ascribed to the mutation from micrococcin sensitivity to resistance. It was also demonstrated that B. subtilis protein BS-L11 is homologous to Escherichia coli ribosomal protein L11. The finding that ribosomes from thiostrepton-resistant mutants do not contain protein L11 suggests that L11 not only is involved in binding of thiostrepton, but also, when mutationally altered, confers resistance to this antibiotic. Although the ribosomes of these strains do not contain protein L11, all thiostrepton-resistant mutants showed the same viability as the parental strain. Thus protein L11 cannot be obligatory for the structure and function of the ribosome.

Anti-Bacterial Agents↗
PubMed1997

Interaction of thiostrepton with an RNA fragment derived from the plastid-encoded ribosomal RNA of the malaria parasite.

Although eukaryotes are not generally sensitive to thiostrepton, growth of the human malaria parasite Plasmodium falciparum is severely inhibited by the drug. The proposed target in P. falciparum is the ribosome of the plastid-like organelle (35 kb circular genome) of unknown function. Positive identification of the drug target would confirm that the organelle is essential for blood-stage development of Plasmodium and help clarify the plastid's biological role. The action of thiostrepton as an antibiotic relates to its affinity for a conserved domain of eubacterial rRNA. Its effect on organelles is unknown. Because a number of different point mutations within the Escherichia coli domain abrogates thiostrepton binding, extensive sequence differences between eubacterial and plastid domains brings into question the site of drug action. We have examined temperature-dependent hyperchromicity profiles of synthetic RNAs corresponding to domains in the plastid and cytoplasmic RNAs of P. falciparum. Thiostrepton induces a tertiary structure in the plastid-like fragment similar to that seen in eubacterial rRNA, even though the two share only about 60% sequence identity. A single point mutation in the plastid-like fragment removes thiostrepton-dependent tertiary structure formation. Thus, the plastid and eubacterial RNAs share a stabilized tertiary structure induced by the drug. This direct indicator of drug sensitivity in eubacteria suggests that the plastid-encoded ribosome is similarly sensitive to thiostrepton and that the plastid is the site of drug action. Correlation of thiostrepton-sensitive and -resistant phenotypes with physical parameters suggests thiostrepton resistance as a selectable marker for plastid transformation.

Animals↗
PubMed1998

The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre.

A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine or threonine. The mutations do not impair either the assembly of the mutant L11 into 70 S ribosomes in vivo or the binding of thiostrepton to ribosomes in vitro. Moreover, the corresponding mutations at proline 22, in a fusion protein of L11 from Escherichia coli with glutathione-S-transferase, did not reduce the binding affinities of the mutated L11 fusion proteins for rRNA of of thiostrepton for the mutant L11-rRNA complexes at rRNA concentrations lower than those prevailing in vivo. Probing the structure of the fusion protein of wild-type L11, from E. coli, using a recently developed protein footprinting technique, demonstrated that a general tightening of the C-terminal domain occurred on rRNA binding, while thiostrepton produced a footprint centred on tyrosine 62 at the junction of the N and C-terminal domains of protein L11 complexed to rRNA. The intensity of this protein footprint was strongly reduced for the mutant L11-rRNA complexes. These results indicate that although, as shown earlier, thiostrepton binds primarily to 23 S rRNA, the drug probably inhibits peptide elongation by impeding a conformational change within protein L11 that is important for the function of the ribosomal GTPase centre. This putative inhibitory mechanism of thiostrepton is critically dependent on proline 18/22. Moreover, the absence of this proline from eukaryotic protein L11 sequences would account for the high thiostrepton resistance of eukaryotic ribosomes.

Amino Acid Sequence↗
PubMed1991

Recognition of the highly conserved GTPase center of 23 S ribosomal RNA by ribosomal protein L11 and the antibiotic thiostrepton.

The antibiotic thiostrepton, a thiazole-containing peptide, inhibits translation and ribosomal GTPase activity by binding directly to a limited and highly conserved region of the large subunit ribosomal RNA termed the GTPase center. We have previously used a filter binding assay to examine the binding of ribosomal protein L11 to a set of ribosomal RNA fragments encompassing the Escherichia coli GTPase center sequence. We show here that thiostrepton binding to the same RNA fragments can also be detected in a filter binding assay. Binding is relatively independent of monovalent salt concentration and temperature but requires a minimum Mg2+ concentration of about 0.5 mM. To help determine the RNA features recognized by L11 and thiostrepton, a set of over 40 RNA sequence variants was prepared which, taken together, change every nucleotide within the 1051 to 1108 recognition domain while preserving the known secondary structure of the RNA. Binding constants for L11 and thiostrepton interaction with these RNAs were measured. Only a small number of sequence variants had more than fivefold effects on L11 binding affinities, and most of these were clustered around a junction of helical segments. These same mutants had similar effects on thiostrepton binding, but more than half of the other sequence changes substantially reduced thiostrepton binding. On the basis of these data and chemical modification studies of this RNA domain in the literature, we propose that L11 makes few, if any, contacts with RNA bases, but recognizes the three-dimensional conformation of the RNA backbone. We also argue from the data that thiostrepton is probably sensitive to small changes in RNA conformation. The results are discussed in terms of a model in which conformational flexibility of the GTPase center RNA is functionally important during the ribosome elongation cycle.

Base Composition↗
PubMed1997

[The isolation and characteristics of mutants of the Saccharopolyspora erythraea strain resistant to thiostrepton].

The formation of thiostreptone resistant spontaneous and nitrosoguanidine-induced mutants in the erythromycin-producing organism Saccharopolyspora erythraea was investigated. The investigated collection of the mutants was heterogeneous by the level of the thiostreptone resistance (2.5 to 20 micrograms/ml). The thiostreptone resistance mutations had a pleiotropic effect: 17 per cent of the mutants was characterized by the growth thermosensitivity and 26 and 5.8 per cent of the mutants were characterized by loss of the ability to form melanine and aerial mycelium respectively. Such phenotypes were most frequent in the mutants resistant to low concentrations of thiostreptone (2 to 5 micrograms/ml). The absolute majority of the isolated thiostreptone resistant mutants was unstable and formed both the antibiotic resistant and the antibiotic sensitive clones. The greatest portion of the strains with high antibiotic activity (20 per cent) was detected among the S. erythraea spontaneous mutants on the medium with 2.5 micrograms/ ml of thiostreptone. It was shown that the instability of the high antibiotic activity in the mutants was associated with loss of the thiostreptone resistance property.

Anti-Bacterial Agents↗
PubMed1996

Cooperative interactions of RNA and thiostrepton antibiotic with two domains of ribosomal protein L11.

Ribosomal protein L11 interacts with a 58-nucleotide domain of large subunit ribosomal RNA; both the protein and its RNA target have been highly conserved. The antibiotic thiostrepton recognizes the same RNA domain, and binds to the ribosome cooperatively with L11. Experiments presented here show that RNA recognition and thiostrepton cooperativity can be attributed to C- and N-terminal domains of L11, respectively. Under trypsin digestion conditions that degrade Bacillus stearothermophilus L11 to small fragments, the target RNA protects the C-terminal 77 residues from digestion, and thiostrepton and RNA in combination protect the entire protein. A 76-residue C-terminal fragment of L11 was overexpressed and shown to fold into a stable structure binding ribosomal RNA with essentially the same properties as full-length L11. An L11.thiostrepton.RNA complex was 100-200-fold more stable than expected on the basis of L11-RNA and thiostrepton-RNA binding affinities; similar measurements with the C-terminal fragment detected no cooperativity with thiostrepton. L11 function is thus more complex than simple interaction with ribosomal RNA; we suggest that thiostrepton mimics some ribosomal component or factor that normally interacts with the L11 N-terminal domain.

Anti-Bacterial Agents↗
PubMed2005

Interactions of the N-terminal domain of ribosomal protein L11 with thiostrepton and rRNA.

Ribosomal protein L11 has two domains: the C-terminal domain (L11-C76) binds rRNA, whereas the N-terminal domain (L11-NTD) may variously interact with elongation factor G, the antibiotic thiostrepton, and rRNA. To begin to quantitate these interactions, L11 from Bacillus stearothermophilus has been overexpressed and its properties compared with those of L11-C76 alone in a fluorescence assay for protein-rRNA binding. The assay relies on 2'-amino-butyryl-pyrene-uridine incorporated in a 58-nucleotide rRNA fragment, which gives approximately 15-fold enhancement when L11 or L11-C76 is bound. Although the pyrene tag weakens protein binding, unbiased protein-RNA association constants were obtained in competition experiments with untagged RNA. It was found that (i) intact B. stearothermophilus L11 binds rRNA with K approximately 1.2 x 10(9) m(-1) in buffers with 0.2 m KCl, about 100-fold tighter than Escherichia coli L11; (ii) the N-terminal domain makes a small, salt-dependent contribution to the overall L11-RNA binding affinity (approximately 8-fold enhancement at 0.2 m KCl), (iii) L11 stimulates thiostrepton binding by 2.3 +/- 0.6 x 10(3)-fold, predicting an overall thiostrepton affinity for the ribosome of approximately 10(9) m(-1), and (iv) the yeast homolog of L11 shows no stimulation of thiostrepton binding. The latter observation resolves the question of why eukaryotes are insensitive to the antibiotic. These measurements also show that it is plausible for thiostrepton to compete directly with EF-G.GDP for binding to the L11-RNA complex, and provide a quantitative basis for further studies of L11 function and thiostrepton mechanism.

Anti-Bacterial Agents↗
PubMed1975

The joining of the 30-S initiation complex with the 50-S subunit, the main target for thiostrepton.

The study undertaken in this paper on the mode of action of thiostrepton provides data which permit a more precise localization of the main target of thiostrepton. There is severe impairment of the joining of the 50-S subunit, probably carrying thiostrepton, with either the 30-S subunit or the 30-S initiation complex. The degree of impairment of this coupling is temperature dependent, being almost completely inhibited at 0 degrees C, whereas at 37 degrees C the effect is much less marked, provided that natural messenger RNA is present. The inhibition of initiation by thiostrepton is more severe in the presence of IF-1, a factor, which similar to thiostrepton, is able to shift the dynamic equilibrium of 70-S in equilibrium 50-S + 30-S more towards dissociation. By means of 14C-labeled IF-2 it is demonstrated that the binding of IF-2 into the 70-S initiation complex is prevented by thiostrepton, which seems to be the main cause for non-coupling.

Anti-Bacterial Agents↗
PubMed1989

Thiostrepton-induced gene expression in Streptomyces lividans.

Thiostrepton induced the expression of four proteins (17, 19, 30, and 56 kilodaltons) of unknown function in Streptomyces lividans. The chromosomal gene which encoded the 19-kilodalton protein (tipA) was cloned and sequenced. Transcription of the tipA promoter was induced at least 200-fold by thiostrepton. The tipA 200-fold by thiostrepton. The tipA transcriptional start site (located by S1 mapping and primer extension experiments) was preceded by a 45-base-pair imperfect inverted-repeat sequence which included the -10 and -35 regions of the promoter. Under noninducing conditions in vivo, this might form a cruciform structure which is not recognized by RNA polymerase. A 143-base-pair fragment including this region was cloned into a promoter probe vector, pIJ486. In this plasmid, pAK114, the thiostrepton-inducible tipA promoter controlled the expression of a kanamycin resistance gene encoding an aminoglycoside phosphotransferase. As little as 1 ng of thiostrepton spotted on a lawn of S. lividans(pAK114) induced kanamycin-resistant growth. Other thiostreptonlike antibiotics also induced tipA, but structurally unrelated antibiotics which inhibit translation had no effect. In S. lividans, the promoter could be induced by thiostrepton during either growth or stationary phase. The tipA promoter should be a valuable tool for expression studies in streptomycetes.

Amino Acid Sequence↗
PubMed1975

Identification of a ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes.

A ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes has been identified using the following criteria: 1. A loss in the thiostrepton binding ability of the ribosome was correlated with the selective removal of ribosomal protein L11. This was achieved by a comparison of the thiostrepton binding ability of 50 S ribosomal subunits treated with 1 M NH4C1 and 50% ethanol at 37 degrees which still contained protein L11, and subunits treated successively at 0 degrees and 37 degrees in the same medium, from which protein L11 had been removed. 2. The thiostrepton binding ability of a ribosomal core containing only seven proteins, produced by treatment of 50 S subunits with 4 M LiC1, was fully restored by the rebinding of protein L11, obtained by Sephadex G-100 fractionation of the 1 M LiC1 split protein fraction from 50 S subunits. In addition, treatment of the 1 M LiC1 split protein fraction with an IgG specific for protein L11, uniquely inhibited the restoration of activity. 3. Thiostrepton binding to the 4 M LiC1 core, reconstituted with the 1 M LiC1 split protein fraction, was blocked by treatment with a monovalent antibody fragment (Fab) prepared against protein L11, but not by treatment with antibodies specific for the proteins of the 4 M LiC1 core. We conclude, therefore, that protein L11 is required for the ribosomal binding of thiostrepton.

Anti-Bacterial Agents↗
PubMed1993

Autogenous transcriptional activation of a thiostrepton-induced gene in Streptomyces lividans.

Although the antibiotic thiostrepton is best known as an inhibitor of protein synthesis, it also, at extremely low concentrations (< 10(-9) M), induces the expression of a regulon of unknown function in certain Streptomyces species. Here, we report the purification of a Streptomyces lividans thiostrepton-induced transcriptional activator protein, TipAL, whose N-terminus is similar to a family of eubacterial regulatory proteins represented by MerR. TipAL was first purified from induced cultures of S.lividans as a factor which bound to and activated transcription from its own promoter. The tipAL gene was overexpressed in Escherichia coli and TipAL protein purified in a single step using a thiostrepton affinity column. Thiostrepton enhanced binding of TipAL to the promoter and catalysed specific transcription in vitro. TipAS, a second gene product of the same open reading frame consisting of the C-terminal domain of TipAL, is apparently translated using its own in-frame initiation site. Since it is produced in large molar excess relative to TipAL after induction and also binds thiostrepton, it may competitively modulate transcriptional activation.

Amino Acid Sequence↗