This laboratory previously showed that truncation of
the gene for RluD, the Escherichia coli pseudouridine
synthase responsible for synthesis of 23S rRNA pseudouridines
1911, 1915, and 1917, blocks pseudouridine formation and
inhibits growth. We now show that RluD mutants at the essential
aspartate 139 allow these two functions of RluD to be separated.
In vitro, RluD with aspartate 139 replaced by threonine
or asparagine is completely inactive. In vivo, the growth
defect could be completely restored by transformation of
an RluD-inactive strain with plasmids carrying genes for
RluD with aspartate 139 replaced by threonine or asparagine.
Pseudouridine sequencing of the 23S rRNA from these transformed
strains demonstrated the lack of these pseudouridines.
Pseudoreversion, which has previously been shown to restore
growth without pseudouridine formation by mutation at a
distant position on the chromosome, was not responsible
because transformation with empty vector under identical
conditions did not alter the growth rate.