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Abstract
The helix spanning nucleotides 1198 to 1247 (helix 1200-1250) in Escherichia coli
23 S ribosomal RNA (rRNA) is functionally important in protein synthesis, and deletions
in this region confer erythromycin resistance. In order to define the structural requirements
for resistance, we have dissected this region using in vitro mutagenesis. Erythromycin
resistance is established after a minimal deletion of three bases, CAU1231 or AUG1232.
The maximum deletion observed to confer resistance is 25 bases. The level of erythromycin
resistance conferred by intermediate sized deletions is variable and some deletion
mutants show a sensitive phenotype. Deletions that extend into the base-pairing between
GCC1208 and GGU1240 result in non-functional 23 S RNAs, which consequently do not
confer resistance. A number of phylogenetically conserved nucleotides have been shown
to be non-essential for 23 S RNA function. However, removal of either these or non-conserved
nucleotides from helix 1200-1250 measurably reduces the efficiency of 23 S RNA in
forming functional ribosomes. We have used chemical probing and a modified primer
extension method to investigate erythromycin binding to wild-type and resistant ribosomes
with a 12-base deletion in 23 S RNA. Erythromycin interacts as strongly with mutant
23 S RNA as with wild-type 23 S RNA. Deletions in the 1200-1250 helix do not therefore
confer resistance by reducing erythromycin binding, but by suppressing the effects
of the drug at the level of its mechanism of action.