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

The upper stem of helix 34, consisting of the basepaired sequences C(1 063)G(1064)U(1065) and A(1191)C(1192)G(1193), is suggested to be invol ved in the binding of spectinomycin. In E.coli 16S rRNA, each of the t hree 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.co li numbering), Since each of these mutations disrupt any of the three basepairs in the upper stem of helix 34, it has been postulated that s pectinomycin can bind to this region and inhibit protein synthesis, on ly if its nucleotides are basepaired. We have tested this hypothesis b y introducing disruptive and compensatory mutations that alter the bas epair G(1064)-C-1192 Using the specialized ribosome system, the transl ational activity of such mutants was determined, in the absence and pr esence of spectinomycin. We show that any of the three disruptive muta tions A(1064), C-1064, and U-1064 confer resistance, in accordance wit h the model for spectinomycin binding. Compensatory mutations A(1064)U (1192), C(1064)G(1192), and U(1064)A(1192), however, maintained the re sistance. This indicates that a basepaired conformation as such is not sufficient for spectinomycin binding, but rather that a G-C pair at p ositions 1064 and 1192 is required. In addition, we find that the tran slational 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 spectinomyc in is present or not. Since the introduction of C(1064)G(1192) is expe cted to increase the stability of the upper stem of helix 34, we sugge st that these mutations impair ribosome function by preventing the (tr ansient) disruption of the upper stem. By analogy, we speculate that s pectinomycin blocks protein synthesis by stabilizing the upper stem. I n both cases, the 30S subunit would be frozen into an inactive conform ation.