SPECTINOMYCIN INTERACTS SPECIFICALLY WITH THE RESIDUES G(1064) AND C(1192) IN 16S RIBOSOMAL-RNA, THEREBY POTENTIALLY FREEZING THIS MOLECULE INTO AN INACTIVE CONFORMATION

The upper stem of helix 34, consisting of the basepaired sequences C(1063)G(1064)U(1065) and A(1191)C(1192)G(1193), is suggested to be involved in the binding of spectinomycin. In E.coli 16S rRNA, each of the three mutations at position C-1192 confers resistance to spectinomycin. In chloroplast ribosomes from tobacco plants and algae, resistance is conferred by single mutations at positions 1064, 1191, and 1193 (E.coli numbering), Since each of these mutations disrupt any of the three basepairs in the upper stem of helix 34, it has been postulated that spectinomycin can bind to this region and inhibit protein synthesis, only if its nucleotides are basepaired. We have tested this hypothesis by introducing disruptive and compensatory mutations that alter the basepair G(1064)-C-1192 Using the specialized ribosome system, the translational activity of such mutants was determined, in the absence and presence of spectinomycin. We show that any of the three disruptive mutations A(1064), C-1064, and U-1064 confer resistance, in accordance with the model for spectinomycin binding. Compensatory mutations A(1064)U(1192), C(1064)G(1192), and U(1064)A(1192), however, maintained the resistance. This indicates that a basepaired conformation as such is not sufficient for spectinomycin binding, but rather that a G-C pair at positions 1064 and 1192 is required. In addition, we find that the translational activity of specialized ribosomes containing the mutations C(1064)G(1192) is 5-fold lower compared to that of ribosomes containing any of the other mutations introduced, regardless whether spectinomycin is present or not. Since the introduction of C(1064)G(1192) is expected to increase the stability of the upper stem of helix 34, we suggest that these mutations impair ribosome function by preventing the (transient) disruption of the upper stem. By analogy, we speculate that spectinomycin blocks protein synthesis by stabilizing the upper stem. In both cases, the 30S subunit would be frozen into an inactive conformation.