2 REGIONS OF THE ESCHERICHIA-COLI 16S RIBOSOMAL-RNA ARE IMPORTANT FORDECODING STOP SIGNALS IN POLYPEPTIDE-CHAIN TERMINATION

Two regions of the 16S rRNA, helix 34, and the aminoacyl site componen t of the decoding site at the base of helix 44, have been implicated i n decoding of translational stop signals during the termination of pro tein synthesis. Antibiotics specific for these regions have been teste d to see how they discriminate the decoding of UAA, UAG, and UGA by th e two polypeptide chain release factors (RF-1 and RF-2). Spectinomycin , which interacts with helix 34, stimulated RF-1 dependent binding to the ribosome and termination. It also stimulated UGA dependent RF-2 te rmination at micromolar concentrations but inhibited UGA dependent RF- 2 binding at higher concentrations. Alterations at position C1192 of h elix 34, known to confer spectinomycin resistance, reduced the binding of f[H-3]Met-tRNA to the peptidyl-tRNA site. They also impaired termi nation in vitro, with both factors and all three stop codons, although the effect was greater with RF-2 mediated reactions. These alteration s had previously been shown to inhibit EF-G mediated translocation. As perturbations in helix 34 effect both termination and elongation reac tions, these results indicate that helix 34 is close to the decoding s ite on the bacterial ribosome. Several antibiotics, hygromycin, neomyc in and tetracycline, specific for the aminoacyl site, were shown to in hibit the binding and function of both RFs in termination with all thr ee stop codons in vitro. These studies indicate that decoding of all s top signals is likely to occur at a similar site on the ribosome to th e decoding of sense codons, the aminoacyl site, and are consistent wit h a location for helix 34 near this site.