title: A ribosomal ATPase is a target for hygromycin B inhibition on Escherichia coli ribosomes.
abstract: We demonstrate that the transfer of fully charged aminoacyl-tRNAs into peptides directed by the MS2 RNA template requires both ATP and GTP, initiation factors (IF1, IF2, and IF3), elongation factors (EF-Tu, EF-Ts, and EF-G), and the ribosomal ATPase (RbbA). The nonhydrolyzable analogue AMPPCP inhibits the reactions, suggesting that hydrolysis of ATP is required for synthesis. The RbbA protein occurs bound to ribosomes and stimulates the ATPase activity of Escherichia coli 70S and 30S particles. The gene encoding RbbA harbors four ATP binding domains; the C-terminal half of the protein bears extensive sequence similarity to EF-3, a ribosome-dependent ATPase. Here, we show that the antibiotic hygromycin B selectively inhibits the ATPase activity of RbbA. Other antibiotics with similar effects on miscoding, streptomycin and neomycin, as well as antibiotics that impair peptide bond synthesis and translocation, had little effect on the ATPase activity of RbbA on 70S ribosomes. Immunoblot analysis indicates that at physiological concentrations, hygromycin B selectively releases RbbA from 70S ribosomes. Hygromycin B protects G1494 and A1408 in the decoding region, and RbbA enhances the reactivity of A889 and G890 of the 16S rRNA switch helix region. Cross-linking and X-ray diffraction data have revealed that this helix switch and the decoding region are in close proximity. Mutations in the switch helix (889-890) region affect translational fidelity and translocation. The binding site of hygromycin B and its known dual effect on the fidelity of decoding and translocation suggest a model for the action of this drug on ribosomes.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, 112 College St., Toronto, Ontario M5G 1L6, Canada. m.ganoza@utoronto.ca
authors.forename: M C
authors.initials: MC
authors.lastname: Ganoza
authors.forename: M C
authors.initials: MC
authors.lastname: Kiel
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001665:Binding Sites; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology; D029968:Escherichia coli Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006921:Hygromycin B / Q000494:pharmacology*; D008958:Models, Molecular; D012270:Ribosomes / Q000187:drug effects* / Q000201:enzymology
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D006921:Hygromycin B; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C422450:RbbA protein, E coli