Phenotypic interactions among mutations in a Thermus thermophilus 16S rRNA gene detected with genetic selections and experimental evolution

Abstract

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.

FIG 1

Locations of mutations identified in this study. (A) Secondary-structure model of 16S rRNA (; http://www.rna.ccbb.utexas.edu/) indicating the sites of mutations identified in this study. Positions of Str^d mutations are indicated by green dots, positions of Strⁱ suppressors by blue dots, and positions of fitness-improving mutations by red dots. Str^r mutations are indicated by pink dots. (B) Positions of mutations in a crystal structure of the 30S subunit bound to streptomycin (PDB entry 4DR3) (35); color codes are as described for panel A, with the addition that the streptomycin binding site is indicated by an orange sphere. (C) Closeup view of the helix 18 pseudoknot region; sites of Str^d mutations in 16S rRNA or ribosomal protein S12 are shown in dark green. Ribosomal proteins S5 and S12 are colored yellow and green, respectively. (D) Sites of mutations located distant from the sites of Str^d mutations. Panels C and D used an apo 30S subunit crystal structure (PDB entry 4DR1) (35).