| Translational accuracy in Saccharomyces cerevisiae is known to be influenced by three small-subunit ribosomal proteins that are conserved in structure and function with their procaryotic counterparts. One of these, encoded by RPS28A and RPS28B (RPS28), is equivalent to bacterial S12. The others, encoded by sup44 (RPS5) or, sup46 and YS11A (RPS13), are homologues of procaryotic S5 and S4, respectively. In Escherichia coli, certain alterations in S12 cause hyperaccurate translation or antibiotic resistance that can be counteracted by other changes in S5 or S4 that reduce translational accuracy or increase antibiotic sensitivity. We show that several changes in RPS28, equivalent to those in E. coli S12, similarly oppose suppressor or antibiotic-sensitive phenotypes associated with SUP44 or SUP46 mutations in yeast, and that one analogous change has an opposite effect. This indicates that RPS28 is a functional, as well as, structural homologue of bacterial S12, but also suggests differences in the ribosomal environments of these proteins. To delineate regions of RPS28 that are critical for its function, we utilized random mutagenesis to show that alterations in RPS28 can diversely affect translational accuracy in yeast. Most changes occur at residues not conserved between procaryotes and eucaryotes, although one is found at a position known to mutate and cause antibiotic sensitivity in bacteria, and another is located in a nominally conserved residue. Certain changes in the amino-terminal part of the protein, which is diverged from the bacterial homologues, cause increased misreading, often corresponding with increased antibiotic sensitivity. Other alterations, found in the well conserved carboxyl-terminal portion, counteract SUP44- or SUP46-associated antibiotic sensitivity. Two of these, appear to specifically antagonize SUP46. We also obtained double-mutants, that show a concerted functional interaction between two suppressor changes at different residues within the amino-terminal portion of RPS28, and analyzed novel dac (diametrical accuracy) mutants, made by combining opposing accuracy mutations in a single allele. The results of our experiments suggest that RPS28 may consist of two distinct functional domains. |
