title: Quantitative binding of antibiotics to ribosomes from a yeast mutant altered on the peptidyl-transferase center.
abstract: Quantitative binding studies of [G-3H]anisomycin and [acetyl-14C]trichodermin to sensitive and resistant 80-S ribosomes from yeasts are described in this work. A single mutation, most probably affecting the ribosome peptidyl transferase centre, appears to have pleiotropic effects on the ribosome leading to resistance to trichodermin and anisomycin and to an increased sensitivity to sparsomycin. Resistance to trichodermin is due to a reduced affinity of ribosomes from the mutant for the antibiotic. Ribosomes from the sensitive strain (Y 1661 bind [acetyl-14C]trichodermin with a dissociation constant of 0.99 muM while those from the resistant one (TR1) bind [acetyl-14C]trichodermin with a dissociation constant of 15.4 muM. Similar results are obtained when the binding of [acetyl-14C]trichodermin to Y 166 and TR1 60-S subunits is studied. The mutant TR1 is also resistant to anisomycin. Although trichodermin and anisomycin bind to the ribosome at mutually exclusive sites, the higher affinity binding of [G-3H]anisomycin that is responsible for the inhibition of the peptidyl transferase center is practically identical for Y 166 and TR1 ribosomes. Therefore, the mutation in the ribosome leading to resistance to trichodermin and anisomycin decreases the affinity for trichodermin but not for anisomycin. Trichodermin, trichothecin and fusarenon X inhibit the binding of [G-3H]anisomycin to TR1 ribosomes to a lower extent than to Y 166 ribosomes, suggesting that the resistance of TR1 ribosomes to the effects of trichothecin and fusarenon X is caused by a decrease in the affinity of the ribosomes for these drugs, as was seen with trichodermin. On the other hand, verrucarin A inhibits [G-3H]anisomycin binding to Y 166 and TR1 ribosomes to a similar extent and therefore its affinity for the ribosome does not appear to be affected by the mutation leading to resistance. Trichothecin, trichodermin and fusarenon X appear to have a common binding site on the 60-S ribosomal subunits, which overlaps or is closely linked to the binding sites of anisomycin and verrucarin A.
mesh_terms: D000217:Acyltransferases / Q000378:metabolism*; D000841:Anisomycin / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D000935:Antifungal Agents / Q000378:metabolism; D001665:Binding Sites; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010458:Peptidyl Transferases / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D011955:Receptors, Drug; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D005656:Fungal Proteins; D012333:RNA, Messenger; D011955:Receptors, Drug; D012269:Ribosomal Proteins; D010649:Phenylalanine; D000841:Anisomycin; D000217:Acyltransferases; D010458:Peptidyl Transferases