Two regions of the Escherichia cqli 16S ribosomal RNA are important for decoding stop signals in polypeptide chain termination

Two regions of the 16S rRNA, helix 34, and the aminoacyl site component of the decoding site at the base of helix 44, have been Implicated In decoding of translational stop signals during the termination of protein synthesis. Antibiotlcs specific for these regions have been tested to see how they discriminate the decoding of UAA, UAG, and UGA by the two polypeptide chain release factors (RF—1 and RF—2). Spectinomycln, which interacts with helix 34, stimulated RF—1 dependent binding to the rlbosome and termination. It also stimulated UGA dependent RF—2 termination at mlcromolar concentrations but inhibited UGA dependent RF—2 binding at higher concentrations. Alterations at position C1192 of helix 34, known to confer spectinomycin resistance, reduced the binding of f[3H]Met-tRNA to the peptldyl-tRNA site. They also impaired termination In vitro, with both factors and all three stop codons, although the effect was greater with RF—2 mediated reactions. These alterations had previously been shown to inhibit EF—G mediated translocatlon. As perturbations In helix 34 effect both termination and elongation reactions, these results indicate that helix 34 Is close to the decoding site on the bacterial rlbosome. Several antibiotics, hygro-mycin, neomycln and tetracycline, specific for the aminoacyl site, were shown to inhibit the binding and function of both RFs in termination with all three stop codons in vitro. These studies Indicate that decoding of all stop signals Is likely to occur at a similar site on the ribosome to the decoding of sense codons, the aminoacyl site, and are consistent with a location for helix 34 near this site.