The overall goal of this research is to determine the functional role of ribosomal RNA (rRNA) in the mechanism of translation. Specific projects have been designed to analyze the details of rRNA molecules from Escherichia coli. These projects will address two related areas: 1) The role of phylogenetically conserved nucleotides in the process of translation. The functional significance of single rRNA nucleotides will be analyzed by site-directed mutagenesis and characterization of mutant function. Specifically, mutations in the 790 region of 16S rRNA will be constructed to explore the role of rRNA in the initiation of protein synthesis. In addition, compensatory mutations involving the same 790 region and the 2755 region of 23S rRNA will test a bimolecular interaction model for ribosomal subunit association. Alterations in the 2660 region of 23S rRNA will also be analyzed to understand how elongation factors interact with rRNA; and 2) The structure and function of tertiary interaction sites within rRNA. Several higher order interactions have been proposed based on phylogenetic comparisons of rRNA sequences. These sites will be tested experimentally by compensatory site-directed mutagenesis. The mutagenic approach will be applied first to proposed pseudoknot and lone pair interactions in the central domain of 16S rRNA. Subsequently, a non-canonical lone pair which is proposed to form in the peptidyl transferase center of 23S rRNA will be mutagenized. Together, the results from these projects will help to define the functional role of rRNA in translation. This in turn will add significantly to our understanding of RNA as a dynamic functional molecule in the cell.
