Effects of mutagenesis of a conserved base-paired site near the decoding region of Escherichia coli 16 S ribosomal RNA.

Eleven of 15 possible single and double mutations were constructed in a cloned copy of Escherichia coli 16 S rDNA at a base-paired site, 1409-1491. Expression of any of these mutations was detrimental to the growth of E. coli. Mutations that substituted unpaired purine bases were lethal in the system described. Otherwise, the degree of detrimental effect on growth-rate was not directly correlated with specific rRNA primary or secondary structures. Using reverse transcription of rRNA isolated from subunits or 70 S ribosomes, we were able to determine the amount of mutant rRNA used in translation. From these experiments, we found that the lethal mutations appeared to be selectively excluded from the pool of 70 S ribosomes following expression from a repressible plasmid. In contrast, a non-lethal mutation was present in subunits, ribosomes and polysomes in approximately equal amounts. Mutations that disrupted base-pairing were found to confer varying levels of resistance to nine aminoglycosides, including four neomycins, two kanamycins, gentamicin, apramycin and hygromycin. A high frequency of reversion from resistant and slow-growth phenotypes due to a host mutation was observed.