Phenotypic Interactions among Mutations in a Thermus thermophilus 16S rRNA Gene Detected with Genetic Selections and Experimental Evolution

During protein synthesis, the ribosome undergoes conformational transitions between functional states, requiring communication between distant structural elements of the ribosome. Despite advances in ribosome structural biology, identifying the protein and rRNA residues governing these transitions remains a significant challenge. Such residues can potentially be identified genetically, given the predicted deleterious effects of mutations stabilizing the ribosome in discrete conformations and the expected ameliorating effects of second-site compensatory mutations. In this study, we employed genetic selections and experimental evolution to identify interacting mutations in the ribosome of the thermophilic bacterium Thermus thermophilus. By direct genetic selections, we identified mutations in 16S rRNA conferring a streptomycin dependence phenotype and from these derived second-site suppressor mutations relieving dependence. Using experimental evolution of streptomycin-independent pseudorevertants, we identified additional compensating mutations. Similar mutations could be evolved from slow-growing streptomycin-resistant mutants. While some mutations arose close to the site of the original mutation in the three-dimensional structure of the 30S ribosomal subunit and probably act directly by compensating for local structural distortions, the locations of others are consistent with long-range communication between specific structural elements within the ribosome.