Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli

There are 10 known putative pseudouridine synthase genes in Escherichia col i. The products of six have been previously assigned, one to formation of t he single pseudouridine in 16S RNA, three to the formation of seven pseudou ridines in 23S RNA, and three to the formation of three pseudouridines in t RNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show th at the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, res pectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseu douridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(IIe1) and tRNA(Asp). Deletion of each of these synthase genes individ ually had no effect on exponential growth in rich media at 25 degreesC, 37 degreesC, or 42 degreesC. A strain lacking RluB and RluF also showed no gro wth defect under these conditions. Mutation of a conserved aspartate in a c ommon sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, al so caused a loss of in vivo activity in all four of the synthases studied i n this work.