A second function for pseudouridine syntheses: A point mutant of RluD unable to form pseudouridines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain

This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli pseudouridine synthase responsible for synthesis of 23S r RNA 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. I n vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with asparta te 139 replaced by threonine or asparagine. Pseudouridine sequencing of the 235 rRNA from these transformed strains demonstrated the lack of these pse udouridines. Pseudoreversion, which has previously been shown to restore gr owth without pseudouridine formation by mutation at a distant position on t he chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.