The primary structure of the rRNA of ribosomes plays a crucial role in protein synthesis. Genetic and biochemical methods are employed to study the relationship between the primary structure and function of selected regions of ribosomal rRNA. Site-specific mutagenesis in M13 is used to make base changes in rRNA. In particular, the focus is on bases in the 530, 790 and 980 loop regions of 16S rRNA of 30S ribosomes of Escherichia coli, which are involved in elongation and tRNA interactions in protein synthesis. The mutated region of rDNA is cloned into one of a variety of plasmids containing the rRNA operon behind constitutive or inducible promoters to permit the study of any type of mutation, including those which are lethal to the cell. To study the properties of a homogeneous population of mutant ribosomes in vivo and to uncover subtle effects of "recessive" mutational changes, mutations are cloned into a 16S rRNA gene which carries the spectinomycin resistant marker (U1192). In the presence of spectinomycin, the cell must rely entirely on plasmid coded 30S ribosomes containing the mutant rRNA. Maxicells are to determine if mutant rRNA is incorporated into subunits, 70S ribosomes and polysomes. Functional effects of mutants are assayed in vitro using defined reactions of protein synthesis.
