Site-directed mutagenesis of ribosomal RNA: Analysis of ribosomal RNA deletion mutants using maxicells☆☆☆

Abstract Until now, a genetic approach to the study of ribosomal RNA (rRNA) has been limited by the absence of mutants coding altered rRNA. In the accompanying paper by Gourse et al. we have described the construction and characterisation of plasmids coding mutant rRNAs. The mutant plasmids each contain a small deletion at one of seven chosen sites in the rRNA coding regions. In order to study the effects of these mutations we have modified the maxicell system of Sancar et al. (1979). We have obtained expression of plasmid-coded rRNA in the complete absence of host-coded rRNA synthesis. Optimal yield and specificity for synthesis of plasmid-coded rRNA were obtained using maxicells generated by use of low ultraviolet light fluences. Under these conditions, maxicells synthesized many proteins with no apparent specificity for expression of only the plasmid-coded polypeptides, despite complete specificity for expression of only the plasmid-coded rRNA. In maxicells containing the wild-type (i.e. unaltered) plasmid, rRNA transcripts were fully processed and assembled into ribosomal subunits. Both 30 S and 50 S subunits assembled in maxicells were matured sufficiently to form 70 S ribosomes. However, small deletions at positions 704 and 1504 in 16 S rRNA abolished the maturation of the mutant precursor 16 S (17 S) rRNAs and concurrently prevented completion of 30 S subunit assembly. Even single-base deletions at two other sites (positions 615 and 1384/1385 in 16 S rRNA) severely retarded both precursor rRNA maturation and 30 S subunit assembly. Mutations at two sites in 23 S rRNA (positions 607 and 1985) had no apparent effect on the processing of the mutant precursor 23 S rRNAs, but partially reduced the production of mature 50 S subunits. Small deletions at position 365 in 23 S rRNA prevented its assembly into 50 S subunits with the capacity to form 70 S ribosomes, although processing of the mutant precursor 23 S rRNA was not prevented.