(G-3H)gougerotin binding to ribosomes. Heterogeneity of eukaryotic ribosomes.

Abstract

The quantitative interaction of [G-3H]gougerotin with Escherichia coli and Saccharomyces cerevisiae ribosomes is studied in this contribution. While E. coli ribosomes show an homogeneous single-site interaction with the antibiotic (dissociation constant = 1.5 μM), S. cerevisiae ribosomes are heterogeneous for [G-3H]gougerotin binding: there is a single-site interaction with dissociation constant = 0.9 μM in 28% of the ribosomes and another single binding site with dissociation constant = 12 μM in 58% of the ribosomes. Antibiotic interaction takes place in all cases with the larger ribosomal subunit. Gougerotin binding is affected by the nature of the ribosomal preparation. Thus, gougerotin has a much stronger affinity for E. coli washed ribosomes than for ribosomes reconstituted from their subunits. This difference is reduced when tRNAphe is prebound, in the presence of poly(U), to the reconstituted ribosomes. Gougerotin is also able to detect differences between the poly(U) · ribosome · Ac-Phe-tRNA complexes formed with either washed or reconstituted ribosomes. The effect of cations, pH, ethanol, temperature and inhibitors of peptide bond formation on [G-3H]gougerotin binding to E. coli and S. cerevisiae ribosomes has also been studied. Only those antibiotics that act on the peptidyl transferase centre of both prokaryotic and eukaryotic ribosomes (blasticidin S, sparsomycin and actinobolin) completely inhibit the binding of [G-3H]-gougerotin. In contrast, griseoviridin strongly enhances [G-3H] gougerotin binding to both types of ribosomes.