Abstract
The gene for RsuA, the pseudouridine synthase that
converts U516 to pseudouridine in 16S ribosomal RNA of
Escherichia coli, has been deleted in strains
MG1655 and BL21/DE3. Deletion of this gene resulted in
the specific loss of pseudouridine516 in both cell lines,
and replacement of the gene in trans on a plasmid
restored the pseudouridine. Therefore, rsuA is
the only gene in E. coli with the ability to produce
a protein capable of forming pseudouridine516. There was
no effect on the growth rate of rsuA−
MG1655 either in rich or minimal medium at either 24, 37,
or 42 °C. Plasmid rescue of the BL21/DE3 rsuA−
strain using pET15b containing an rsuA gene with
aspartate102 replaced by asparagine or threonine demonstrated
that neither mutant was active in vivo. This result supports
a role for this aspartate, located in a unique GRLD sequence
in this gene, at the catalytic center of the synthase.
Induction of wild-type and the two mutant synthases in
strain BL21/DE3 from genes in pET15b yielded a strong overexpression
of all three proteins in approximately equal amounts showing
that the mutations did not affect production of the protein
in vivo and thus that the lack of activity was not due
to a failure to produce a gene product. Aspartate102 is
found in a conserved motif present in many pseudouridine
synthases. The conservation and distribution of this motif
in nature was assessed.
