BASE CHANGES AT POSITION-792 OF ESCHERICHIA-COLI 16S RIBOSOMAL-RNA 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.