Previous work from this laboratory (Nurse et al.,
RNA, 1995, 1:102–112) established
that TruB, a pseudouridine (Ψ) synthase from Escherichia
coli, was able to make Ψ55 in tRNA transcripts
but not in transcripts of full-length or fragmented 16S
or 23S ribosomal RNAs. By deletion of the truB
gene, we now show that TruB is the only protein in E.
coli able to make Ψ55 in vivo. Lack of TruB and
Ψ55 did not affect the exponential growth rate but
did confer a strong selective disadvantage on the mutant
when it was competed against wild-type. The negative selection
did not appear to be acting at either the exponential or
stationary phase. Transformation with a plasmid vector
conferring carbenicillin resistance and growth in carbenicillin
markedly increased the selective disadvantage, as did growth
at 42 °C, and both together were approximately additive
such that three cycles of competitive growth sufficed to
reduce the mutant strain to ∼0.2% of its original value.
The most striking finding was that all growth effects could
be reversed by transformation with a plasmid carrying a
truB gene coding for a D48C mutation in TruB.
Direct analysis showed that this mutant did not make Ψ55
under the conditions of the competition experiment. Therefore,
the growth defect due to the lack of TruB must be due to
the lack of some other function of the protein, possibly
an RNA chaperone activity, but not to the absence of Ψ55.