Base changes at position 792 of Escherichia coli 16S rRNA affect assembly of 70S ribosomes

To investigate the function of base 792 of 16S rRNA in 30S ribosomes of Escherichia coli, the wild-type (adenine) residue was changed to guanine, cytosine, or uracil by oligonucleotide-directed mutagenesis. Each base change conferred a unique phenotype on the cells. Cells containing plasmid pKK3535 with G⁷⁹² or T⁷⁹² showed no difference in generation time in LB broth containing ampicillin, whereas cells with C⁷⁹² exhibited a 20% increase in generation time in this medium. To study the effect on cell growth of a homogeneous population of mutant ribosomes, the mutations were cloned into the 16S rRNA gene on pKK3535 carrying a spectinomyein-resistance marker (thymine at position 1192), and the cells were grown with spectinomycin. Cells containing G⁷⁹² or C⁷⁹² showed 16% and 56% increases in generation time, respectively, and a concomitant decrease in ³⁵S assimilation into proteins. Cells with T⁷⁹² did not grow in spectinomycin-containing medium. Maxicell analyses indicated decreasing ability to form 70S ribosomes from 30S subunits containing guanine, cytosine, or uracil at position 792 in 16S rRNA. It appeared that C⁷⁹²-containing 30S ribosomes had lost the ability to bind initiation factor 3.