Selective perturbation of G530 of 16 S rRNA by translational miscoding agents and a streptomycin-dependence mutation in protein S12

Abstract

Previous studies have shown that a concise set of universally conserved bases in 16 S rRNA are strongly protected from attack by chemical probes when tRNA is bound specifically to the ribosomal A site. Two of these bases, A1492 and A1493, are located in the cleft of the 30 S subunit, the site of codon-anticodon interaction. A third residue, G530, is located within the highly conserved 530 stem-loop, a region that is involved in interactions with proteins S4 and S12, mutations in which perturb the translational error frequency. The 530 loop is also thought to be located at or near the site of interaction of elongation factor Tu on the 30 S subunit, a location that is distinct from the decoding site. This study monitors the response of these two A-site-related regions of 16 S rRNA to a variety of translational miscoding agents. Several of these agents, including streptomycin, neomycin and ethanol, selectively potentiate tRNA-dependent protection of residue G530 from kethoxal modification; in contrast, little change in reactivity of residues A1492 and A1493 is observed. These results are consistent with the previously demonstrated importance of G530 for A-site function and, moreover, suggest a common mechanism of action for these miscoding agents, even though they appear to have distinctly different modes of interaction with 16 S rRNA. In contrast to the miscoding agents, we find that a streptomycin-dependence (SmD) mutation in protein S12, which causes ribosomes to be hyperaccurate, antagonizes tRNA-dependent protection of G530. The possibility that 5' or 3' flanking regions of mRNA could be involved in tRNA-dependent protection of G530 was tested by using different lengths of oligo(U) to promote binding of tRNA(Phe) to the A site. The relative levels of protection of G530, A1492 and A1493 were unchanged as the size of the mRNA fragment was decreased from 16 to 6 bases in length. We conclude, therefore, that for protection of G530 to be the result of direct contact with message, it must necessarily be located directly at the decoding site; otherwise, its protection is best explained by allosteric interactions, either with mRNA, or with the codon-anticodon complex. These results are discussed in terms of a model wherein the conformation of the 530 loop is correlated with the affinity of the ribosome for elongation factor Tu.