We will use genetic approaches to study the structure, function and assembly of ribosomal RNA in E. coli. The main feature of the method is to use antibiotic-resistance mutations in 16S and 23S rRNA as genetic markers. This allows us to use both random and site-directed mutagenesis approaches. The construction of inducible rRNA operons allows us to identify lethal mutations, as well. Mutations that confer a variety of phenotypes, including temperature-sensitive, cold-sensitive, "downs," nulls, Ram, suppressors, etc. will be isolated and localized by marker rescue and DNA sequencing. Second site revertants will also be sought, in an effort to identify long-range structural and functional interactions involving ribosomal RNA.
