Constructing mutant ribosomes containing mutant ribosomal RNAs

The ribosome is the factory for protein biosynthesis, consisting of 3 different ribosomal RNA (rRNA) molecules (16S, 23S, and 5S rRNAs in prokaryotes) and more than 50 different ribosomal proteins. Because almost all organisms have multiple operons for rRNA genes (rrn operons), mutational analysis of ribosomes has inevitable technical difficulties, particularly for analyzing the functions of the 16S and 23S rRNAs, which form part of the core structure for the small (30S) and large (50S) subunits, respectively. In this chapter, we introduce six major strategies that allow researchers to perform mutational studies of the prokaryotic ribosome, particularly by focusing on the analysis of the 16S and 23S rRNA molecules. Although conventional mutational studies allow only for a small number of nucleotide changes simultaneously, recent approach developed by our group circumvents this problem in the Escherichia coli 16S rRNA gene, allowing for changes of up to 20% of the total nucleotides by interspecies exchange of the gene with that from foreign (non-E. coli) bacteria. The outcome of this novel technique has led to the discovery of an unexpected, nontranslational function (ribonuclease inhibitor) in the 16S rRNA molecule. The introduction of such a large sequence perturbation into the central core of the ribosome will open up a new era of ribosomal engineering to create highly functional ribosomes or phenotypic improvements of the host cell, which would be advantageous for biotechnological applications.