The goal of this proposal is to investigate structure/function relationships of eukaryotic ribosomal RNA(rRNA). An extensive mutational analysis of eukaryotic rRNA will be undertaken for the first time. The focus will be on elements of rRNA important for translational fidelity. A novel yeast genetic system, devised by E. Morgan, that makes it possible to obtain strains with a pure population of mutant rRNA despite the highly repeated nature of eukaryotic rDNA genes, has been adapted for use in this study. This system has technical advantages even over E. coli systems for the study of rDNA mutations. The principal investigator's considerable experience isolating and analyzing mutations that affect translational fidelity, and her success with the Morgan system, make her uniquely qualified for this work. Site-directed and randomly induced rDNA mutations will be screened for affects on drug sensitivity, readthrough of stop-codons and reading frame shifts. The sequences and phenotypes of these mutations will test specific hypotheses that base pairing between rRNA and mRNA or tRNA is involved in various aspects of translation. Intragenic suppressors of rDNA mutations will identify interactions between different regions of the rRNA. Mechanisms of action of some of these mutations will be explored with collaborators e.g. by testing for affects on ribosomal binding of elongation factors. Some of the yeast mutations to be studied here correspond to known E. coli rRNA mutations and will indicate which of the E. coli results reflect fundamental principles of protein synthesis that are conserved in all ribosomes. More importantly, the proposed studies will uncover mutations in conserved rRNA regions, not yet found in E. coli or any other organism and will lead to a new understanding of rRNA structure/function elements common to prokaryotes and eukaryotes. Indeed, one such new mutation in the highly conserved ricin loop was obtained during the preliminary studies for this proposal. Finally, the random mutagenesis screens should also reveal information that is specific to eukaryotic ribosomes. It has been suggested that rRNA is the active component of the ribosome and that the role of r-proteins is to influence the rRNA conformation. The relationship between rRNA and r-proteins will be investigated by selecting for mutations in 185 rRNA that mimic or suppress the phenotypes of known translational-ambiguity mutations in r-proteins. Collaborators will test the effects of some of these rRNA mutations on l) the ability of 185 rRNA to bind r-protein 513, and 2) the conformation of rRNA in the mutant ribosomes. If phenotypically identical ribosomes have the same rRNA conformational changes whether they contain mutations in rRNA or r- protein, the hypothesis that the function of r-proteins is to adjust the rRNA conformation would be supported. This hypothesis will also be tested by trying to isolate rRNA mutations that cause the same conformational change normally provided by the wild-type 513 r-protein. Accordingly, mutations in 185 rRNA that compensate for the loss of the essential S 13 r-protein will be sought.
