We propose to genetically identify RNA-RNA interactions contributing to the subunit interface of the ribosome. We will construct 16S rRNA alleles predicted to confer defects in subunit association. These mutant 30S subunits will also contain an altered anti-Shine-Dalgarno region (denoted ASD*), allowing us to measure specific translational activity in vivo while circumventing problems such as recessive and dominant lethal 16S alleles. Using ASD*, we will develop a quick chromatography-based method to assay subunit association in vitro. With a subset of strains expressing association-defective 30S subunits, we will select second site suppressors which restore translational activity. These suppressor mutations may map to 23S rRNA and identify intermolecular RNA-RNA contacts. Insights gained by this study may (1) provide additional constraints for modeling ribosome structure in three dimensions, (2) aid design of structural studies of functional rRNA domains, and (3) provide tools to study initiation and elongation mechanisms.
| Fredrick, Kurt; Noller, Harry F (2002) Accurate translocation of mRNA by the ribosome requires a peptidyl group or its analog on the tRNA moving into the 30S P site. Mol Cell 9:1125-31 |
| Fredrick, K; Dunny, G M; Noller, H F (2000) Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30 S subunits. J Mol Biol 298:379-94 |
