Functional effect of deletion and mutation of the Escherichia coli ribosomal RNA and tRNA pseudouridine synthase RluA

J Biol Chem. 1999 Jul 2;274(27):18880-6. doi: 10.1074/jbc.274.27.18880.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Intramolecular Transferases / genetics*
  • Intramolecular Transferases / metabolism
  • Kinetics
  • Mutagenesis
  • Pseudouridine / genetics
  • RNA, Ribosomal, 23S / genetics*
  • RNA, Ribosomal, 23S / metabolism
  • Sequence Deletion

Substances

  • RNA, Ribosomal, 23S
  • Pseudouridine
  • Intramolecular Transferases
  • pseudouridine synthases