A pseudouridine synthase required for the formation of two universally conserved pseudouridines in ribosomal RNA is essential for normal growth of Escherichia coli.
Escherichia coli rRNA contains 10 pseudouridines of unknown function. They are made by synthases, each of which is specific for one or more pseudouridines. Here we show that the sfhB (yfil) ORF of E. coli is a pseudouridine synthase gene by cloning, protein overexpression, and reaction in vitro with rRNA transcripts. Gene disruption by miniTn10(cam) insertion revealed that this synthase gene, here renamed rluD, codes for a synthase which is solely responsible in vivo for synthesis of the three pseudouridines clustered in a stem-loop at positions 1911, 1915, and 1917 of 23S RNA. The absence of RluD results in severe growth inhibition. Both the absence of pseudouridine and the growth defect could be reversed by insertion of a plasmid carrying the rluD gene into the mutant cell, clearly linking both effects to the absence of RIuD. This is the first report of a major physiological defect due to the deletion of any pseudouridine synthase. Growth inhibition may be due to the lack of one or more of the 23S RNA pseudouridines made by this synthase since pseudouridines 1915 and 1917 are universally conserved and are located in proximity to the decoding center of the ribosome where they could be involved in modulating codon recognition.
Raychaudhuri
S
S
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
Conrad
J
J
Hall
B G
BG
Ofengand
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002455:Cell Division; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics*; D020043:Genes, Essential / Q000235:genetics; D005816:Genetic Complementation Test; D006836:Hydro-Lyases; D019751:Intramolecular Transferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011560:Pseudouridine / Q000096:biosynthesis*; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017423:Sequence Analysis, RNA
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D011560:Pseudouridine; D006836:Hydro-Lyases; C115603:RluD protein, E coli; D019751:Intramolecular Transferases