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The influence of base pairing in the penultimate stem of Escherichia coli 16S rRNA (defined as nt 1409-1491) on ribosome function has been addressed by the construction of mutations in this region of rRNA. Two sets of mutations were made on either side of a structurally conserved region in the penultimate stem that disrupted base pairing, while a third set of mutations replaced the wild-type sequence with other base pair combinations. The effects of these mutations were analyzed in vivo and in vitro . The mutations that disrupted base pairing caused significant increases in cell doubling times as well as a severe subunit association defect and a modest increase in frame shifting and stop codon read-through. Restoration of base pairing restored wild-type growth rates, decoding and subunit association, indicating that base pairing in this region is essential for proper ribosome function.

The importance of base pairing in the penultimate stem of Escherichia coli 16S rRNA for ribosomal subunit association