title: Functional effect of deletion and mutation of the Escherichia coli ribosomal RNA and tRNA pseudouridine synthase RluA.
abstract: The Escherichia coli gene rluA, coding for the pseudouridine synthase RluA that forms 23 S rRNA pseudouridine 746 and tRNA pseudouridine 32, was deleted in strains MG1655 and BL21/DE3. The rluA deletion mutant failed to form either 23 S RNA pseudouridine 746 or tRNA pseudouridine 32. Replacement of rluA in trans on a rescue plasmid restored both pseudouridines. Therefore, RluA is the sole protein responsible for the in vivo formation of 23 S RNA pseudouridine 746 and tRNA pseudouridine 32. Plasmid rescue of both rluA- strains using an rluA gene carrying asparagine or threonine replacements for the highly conserved aspartate 64 demonstrated that neither mutant could form 23 S RNA pseudouridine 746 or tRNA pseudouridine 32 in vivo, showing that this conserved aspartate is essential for enzyme-catalyzed formation of both pseudouridines. In vitro assays using overexpressed wild-type and mutant synthases confirmed that only the wild-type protein was active despite the overexpression of wild-type and mutant synthases in approximately equal amounts. There was no difference in exponential growth rate between wild-type and MG1655(rluA-) either in rich or minimal medium at 24, 37, or 42 degrees C, but when both strains were grown together, a strong selection against the deletion strain was observed.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, USA.
authors.forename: S
authors.initials: S
authors.lastname: Raychaudhuri
authors.forename: L
authors.initials: L
authors.lastname: Niu
authors.forename: J
authors.initials: J
authors.lastname: Conrad
authors.forename: B G
authors.initials: BG
authors.lastname: Lane
authors.forename: J
authors.initials: J
authors.lastname: Ofengand
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D019751:Intramolecular Transferases / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D011560:Pseudouridine / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D012338:RNA, Ribosomal, 23S; D011560:Pseudouridine; D019751:Intramolecular Transferases; C096995:pseudouridine synthases