Deletion of the Escherichia coli pseudouridine synthase gene truB blocks formation of pseudouridine 55 in tRNA in vivo, does not affect exponential growth, but confers a strong selective disadvantage in competition with wild-type cells

Previous work from this laboratory (Nurse et al., RNA, 1995, 1:102-112) est ablished that TruB, a pseudouridine (Psi) synthase from Escherichia coli, w as able to make Psi 55 in tRNA transcripts but not in transcripts of full-l ength 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 Psi 55 in vivo. Lack of TruB and Psi 55 did not affect the exponential growth rate but did confer a strong selective disadvantage on the mutant when it was c ompeted against wild-type. The negative selection did not appear to be acti ng at either the exponential or stationary phase. Transformation with a pla smid vector conferring carbenicillin resistance and growth in carbenicillin markedly increased the selective disadvantage, as did growth at 42 degrees C, and both together were approximately additive such that three cycles of competitive growth sufficed to reduce the mutant strain to similar to0.2% o f its original value. The most striking finding was that all growth effects could be reversed by transformation with a plasmid carrying a truB gene co ding for a D48C mutation in TruB. Direct analysis showed that this mutant d id not make Psi 55 under the conditions of the competition experiment. Ther efore, 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, bu t not to the absence of Psi 55.