title: The allosteric three-site model for the ribosomal elongation cycle. New insights into the inhibition mechanisms of aminoglycosides, thiostrepton, and viomycin.
abstract: According to the allosteric three-site model for the ribosomal elongation cycle (Rheinberger, H.J. and Nierhaus, K.H. (1986) J. Biol. Chem. 261, 9133-9139), two types of A site (aminoacyl-tRNA site) occupation exist. First is the A site occupation after initiation (i-type), with only one site, the P site (peptidyl-tRNA site), being prefilled with a tRNA (initiator tRNA). Second is the A site occupation after an elongation cycle (e-type), with two prefilled sites, namely the P and E sites containing peptidyl-tRNA and deacylated tRNA, respectively. The individual reactions of the elongation cycle were tested, including both types of A site occupation in the presence of various antibiotics. A test system was used allowing the functional studies to be made with quantitative tRNA binding at 6 mM Mg2+. The following results were obtained: 1) thiostrepton (5 x 10(-6) M) induced a complete block of both EF-(elongation factor) G dependent and EF-G independent translocation, in agreement with older observations. The A-site occupation of the e-type was severely inhibited in contrast to that of the i-type. Thus, thiostrepton blocks the allosteric transitions in both directions, i.e. the transition from pre- to post-translocational state (translocation) and that from the post- to the pre-translocational state (A site occupation of the e-type). In addition the ribosomal binding of EF-G.[3H] GMPPNP was inhibited by about 60%. 2) Similarly, viomycin (5 x 10(-5) M) appears to be an inhibitor of both allosteric transitions, since it strongly inhibited the e-type (but not the i-type) A site occupation in addition to translocation. 3) The aminoglycosides streptomycin, hygromycin B, neomycin, kanamycin, and gentamicin prevented A site occupation of the e-type (residual activity below 15%). Neomycin and hygromycin, in addition, blocked the translocation reaction. Only marginal effects were observed with A site occupation of the i-type. It appears that the inhibition of the A site binding of the e-type (allosteric transition from the post- to the pretranslocational state) is the predominant effect of the misreading-inducing aminoglycosides.
authors.affiliation: Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin-Dahlem, West Germany.
authors.forename: T P
authors.initials: TP
authors.lastname: Hausner
authors.forename: U
authors.initials: U
authors.lastname: Geigenmüller
authors.forename: K H
authors.initials: KH
authors.lastname: Nierhaus
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D005700:Galactosyltransferases / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D008034:Lincomycin / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D008957:Models, Genetic; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects*; D013883:Thiostrepton / Q000494:pharmacology*; D014756:Viomycin / Q000494:pharmacology*
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D012345:RNA, Transfer, Amino Acid-Specific; D012360:RNA, Transfer, Phe; D006165:Guanylyl Imidodiphosphate; D008034:Lincomycin; D005700:Galactosyltransferases; C023235:glucosylceramide beta-1-4-galactosyltransferase; D013883:Thiostrepton; D008274:Magnesium; D014756:Viomycin