-
The Journal of Biological Chemistry Nov 2003Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S...
Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site.
Topics: Dose-Response Relationship, Drug; Escherichia coli; Fusidic Acid; Guanosine Triphosphate; Hydrolysis; Kinetics; Models, Biological; Mutation; Peptide Elongation Factor G; Protein Binding; Protein Transport; RNA, Messenger; RNA, Transfer; Ribosomes; Thiostrepton; Time Factors; Viomycin
PubMed: 12960150
DOI: 10.1074/jbc.M304834200 -
Antimicrobial Agents and Chemotherapy Sep 2003The tuberactinomycin antibiotics are essential components in the drug arsenal against Mycobacterium tuberculosis infections and are specifically used for the treatment...
The tuberactinomycin antibiotics are essential components in the drug arsenal against Mycobacterium tuberculosis infections and are specifically used for the treatment of multidrug-resistant tuberculosis. These antibiotics are also being investigated for their targeting of the catalytic RNAs involved in viral replication and for the treatment of bacterial infections caused by methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant enterococci. We report on the isolation, sequencing, and annotation of the biosynthetic gene cluster for one member of this antibiotic family, viomycin, from Streptomyces sp. strain ATCC 11861. This is the first gene cluster for a member of the tuberactinomycin family of antibiotics sequenced, and the information gained can be extrapolated to all members of this family. The gene cluster covers 36.3 kb of DNA and encodes 20 open reading frames that we propose are involved in the biosynthesis, regulation, export, and activation of viomycin, in addition to self-resistance to the antibiotic. These results enable us to predict the metabolic logic of tuberactinomycin production and begin steps toward the combinatorial biosynthesis of these antibiotics to complement existing chemical modification techniques to produce novel tuberactinomycin derivatives.
Topics: Amino Acids; Antibiotics, Antitubercular; Arginine; Chromatography, High Pressure Liquid; Cosmids; DNA, Bacterial; Enviomycin; Gene Library; Lysine; Multigene Family; Oligopeptides; Streptomyces; Viomycin
PubMed: 12936980
DOI: 10.1128/AAC.47.9.2823-2830.2003 -
The EMBO Journal May 2002Ribosome recycling factor (RRF) together with elongation factor G (EF-G) disassembles the post- termination ribosomal complex. Inhibitors of translocation, thiostrepton,...
Ribosome recycling factor (RRF) together with elongation factor G (EF-G) disassembles the post- termination ribosomal complex. Inhibitors of translocation, thiostrepton, viomycin and aminoglycosides, inhibited the release of tRNA and mRNA from the post-termination complex. In contrast, fusidic acid and a GTP analog that fix EF-G to the ribosome, allowing one round of tRNA translocation, inhibited mRNA but not tRNA release from the complex. The release of tRNA is a prerequisite for mRNA release but partially takes place with EF-G alone. The data are consistent with the notion that RRF binds to the A-site and is translocated to the P-site, releasing deacylated tRNA from the P- and E-sites. The final step, the release of mRNA, is accompanied by the release of RRF and EF-G from the ribosome. With the model post-termination complex, 70S ribosomes were released from the post-termination complex by the RRF reaction and were then dissociated into subunits by IF3.
Topics: Escherichia coli; Macromolecular Substances; Peptide Chain Termination, Translational; Peptide Elongation Factor G; Protein Synthesis Inhibitors; Proteins; RNA, Messenger; RNA, Transfer; Ribosomal Proteins; Ribosomes
PubMed: 11980724
DOI: 10.1093/emboj/21.9.2272 -
The Journal of Antibiotics Nov 1999Two cyclic homopentapeptides, CP-101,680 and CP-163,234 [6a-(3',4'-dichlorophenylamino) analogs of viomycin and capreomycin, respectively], were identified as novel...
Two cyclic homopentapeptides, CP-101,680 and CP-163,234 [6a-(3',4'-dichlorophenylamino) analogs of viomycin and capreomycin, respectively], were identified as novel antibacterial agents for the treatment of animal disease, especially for livestock respiratory disease. The in vitro microbiological characterization of both CP-101,680 and CP-163,234 was carried out using their parent compounds, viomycin and capreomycin, as controls. This characterization included antibacterial spectrum, influence of media, inoculum size, pH, EDTA, polymixin B nonapeptide (PMBN), serum, cell-free protein synthesis inhibition, and time-kill kinetics. Our results indicated that the capreomycin analog, CP-163,234, showed slightly improved in vitro potency over the viomycin analog, CP-101,680. Both analogs showed very potent cell-free protein synthesis inhibition activity and were bactericidal against Pasteurella haemolytica, P. multocida and Actinobacillus pleuropneumoniae at the level of 4 times and 8 times MICs. CP-163,234 was bactericidal at the level of 4x and 8x MIC against E. coli, but re-growth was observed after 24 hours incubation at both concentrations of CP-101,680.
Topics: Animal Diseases; Animals; Anti-Bacterial Agents; Bacteria; Capreomycin; Culture Media; Edetic Acid; Humans; Hydrogen-Ion Concentration; Microbial Sensitivity Tests; Polymyxin B; Viomycin
PubMed: 10656573
DOI: 10.7164/antibiotics.52.1007 -
The EMBO Journal Jan 2000One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the... (Review)
Review
One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science.
Topics: Aminoglycosides; Animals; Anti-Bacterial Agents; Drug Design; Humans; Models, Molecular; Nucleic Acid Conformation; Protein Biosynthesis; RNA; RNA-Binding Proteins; Viomycin
PubMed: 10619838
DOI: 10.1093/emboj/19.1.1 -
European Journal of Biochemistry Jan 1999In Escherichia coli, 4.5S RNA is found in complexes with both protein translocation protein, Ffh (a bacterial homolog of mammalian SRP54) and protein synthesis...
In Escherichia coli, 4.5S RNA is found in complexes with both protein translocation protein, Ffh (a bacterial homolog of mammalian SRP54) and protein synthesis elongation factor G (EF-G). To analyze the function of 4.5S RNA in translation, we initially assessed the sensitivity of the association of 4.5S RNA with the ribosome after treatment with antibiotics that affect various stages of protein synthesis. Fusidic acid and viomycin caused 4.5S RNA to cosediment with the 70S ribosomal fraction, indicating that 4.5S RNA enters the ribosome before ribosomal translocation and release of EF-G-GDP from the ribosome. On the other hand, depletion of 4.5S RNA led to the retention of a significant amount of EF-G on 70S ribosomes. In addition, 4.5S RNA shares a conserved decanucleotide sequence (58GAAGCAGCCA67) motif with the characterized EF-G-binding site at positions 1068-1077 on 23S RNA. We therefore examined by gel mobility-shift assay whether or not mutations in the domain-IV region of 4.5S RNA, including this conserved motif, disturb the binding of EF-G to 23S RNA. Any mutation at the C62, G64 or A67 residues within this motif abolished competition activity. Therefore, we propose that 4.5S RNA is concerned with the mode of association of EF-G with the ribosomes. Moreover, this function depends on the secondary structure of 4.5S RNA as well as a ten-base sequence conserved between the two RNAs.
Topics: Bacterial Proteins; Base Sequence; Binding Sites; Binding, Competitive; Centrifugation, Density Gradient; Conserved Sequence; Escherichia coli; Fusidic Acid; Guanosine Diphosphate; Mutation; Nucleic Acid Conformation; Peptide Elongation Factor G; Peptide Elongation Factors; Protein Biosynthesis; Puromycin; RNA, Bacterial; RNA, Ribosomal, 23S; Ribosomes; Signal Recognition Particle; Tetracycline; Viomycin
PubMed: 9914538
DOI: 10.1046/j.1432-1327.1999.00077.x -
Journal of Bacteriology Jan 1998An Escherichia coli mutant lacking the modified nucleotide m1G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83-100, 1970). In...
An Escherichia coli mutant lacking the modified nucleotide m1G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83-100, 1970). In this study, we localize the position of the m1G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. The rrmA gene encoding 23S rRNA m1G745 methyltransferase was mapped to bp 1904000 on the E. coli chromosome and identified to be identical to the previously sequenced gene yebH.
Topics: Chromatography, High Pressure Liquid; Drug Resistance, Microbial; Escherichia coli; Genetic Complementation Test; Methyltransferases; Microbial Sensitivity Tests; Mutation; Open Reading Frames; RNA, Ribosomal, 23S; Viomycin
PubMed: 9440525
DOI: 10.1128/JB.180.2.359-365.1998 -
The Journal of Antibiotics Aug 1997
Topics: Cysteine; Enviomycin; Fermentation; Protein Synthesis Inhibitors; Streptomyces
PubMed: 9315086
DOI: 10.7164/antibiotics.50.698 -
Journal of Bacteriology Aug 1997We examined the molecular mechanisms of resistance to kanamycin and viomycin in Mycobacterium smegmatis. All of the M. smegmatis strains with high-level kanamycin...
We examined the molecular mechanisms of resistance to kanamycin and viomycin in Mycobacterium smegmatis. All of the M. smegmatis strains with high-level kanamycin resistance had a nucleotide substitution from A to G at position 1389 of the 16S rRNA gene (rrs). This position is equivalent to position 1408 of Escherichia coli, and mutation at this position is known to cause aminoglycoside resistance. Mutations from G to A or G to T at position 1473 of the M. smegmatis rrs gene were found in viomycin-resistant mutants which had been designated vicB mutants in our earlier studies. Using the M. smegmatis conjugation system, we confirmed that these mutations indeed contributed to kanamycin and viomycin resistance, and kanamycin susceptibility was dominant over resistance in a heterogenomic strain. Additional experiments showed that three of four Mycobacterium tuberculosis strains with high-level kanamycin resistance had a mutation from A to G at position 1400, which was equivalent to position 1389 of M. smegmatis.
Topics: Base Sequence; Capreomycin; Conjugation, Genetic; DNA, Bacterial; Drug Resistance, Microbial; Drug Resistance, Multiple; Genes, Dominant; Genes, Recessive; Genome, Bacterial; Kanamycin Resistance; Molecular Sequence Data; Mutation; Mycobacterium; Mycobacterium tuberculosis; RNA, Bacterial; RNA, Ribosomal, 16S; Sequence Analysis, RNA; Viomycin
PubMed: 9244267
DOI: 10.1128/jb.179.15.4795-4801.1997 -
Microbiology (Reading, England) Mar 1997A system for gene disruption and replacement based on a streptomycete temperate phage vector was developed to introduce DNA in the rapamycin-producing Streptomyces...
A system for gene disruption and replacement based on a streptomycete temperate phage vector was developed to introduce DNA in the rapamycin-producing Streptomyces hygroscopicus strain ATCC 29253. This will be useful in attempts to produce, through genetic manipulation, novel forms of the therapeutically important immunosuppressive drug rapamycin. Recombinant phages were constructed from the phi C31 phage derivative KC515 (C+ attp) carrying a thiostrepton or viomycin resistance gene along with segments of the S. hygroscopicus chromosome. Each of the cloned segments also contained the aphll neomycin/kanamycin resistance gene to enable gene replacement by loss of the phage-derived DNA. Specific deletion of the entire polyketide synthase (PKS) believed to govern rapamycin biosynthesis resulted in the loss of rapamycin production. In contrast, disruption or deletion of a region predicted to encode four PKS open reading frames, or another region predicted to encode another PKS plus a cytochrome P450 hydroxylase and ferredoxin, had no effect on the production of rapamycin or nigericin, a polyether antibiotic also produced by S. hygroscopicus. Therefore, S. hygroscopicus may have the capacity to produce polyketides additional to rapamycin and nigericin.
Topics: Bacteriophages; Cloning, Molecular; DNA, Bacterial; DNA, Recombinant; DNA, Viral; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Polyenes; Sirolimus; Streptomyces
PubMed: 9084171
DOI: 10.1099/00221287-143-3-875