Binding of Thiostrepton to the Ribosomes of Escherichia coli: Characterization and Stoichiometry of Binding

Abstract

An improved procedure for the production and isolation of 35S-labelled thiostrepton is described. The product was characterized as thiostrepton by comparison with non-radioactive thiostrepton in terms of mobility in thin-layer chromatography, inhibition of growth of sensitive organisms. inhibition of ribosome. EF-G. GDP complex formation and by isotope dilution of binding to of either non-radioactive or [35S]thiostrepton was required. Also, the addition of 2 pmol of [35S]-thiostrepton was required for the maximum binding of 1 pmol of thiostrepton to 1 pmol of ribosomes. Together, these observations suggested that an equal amount of an inactive variant is present in both preparations. The characteristics of the binding of the [35S]thiostrepton to ribosomes were examined. The binding was rapid and relatively independent of pH or NH+4 or Mg2+ concentrations. It was specific for the 50-S ribosomal subunit, inhibited by the presence of pre-bound EF-G, and species specific for prokaryotes. Quantitation of the binding was consistent with one binding site per ribosome. Taken together, these characteristics correspond to those which would be predicted from the previously known characteristics of the inhibitory activity.