Molecular Determinants for 23S rRNA Recognition and Modification by the E. coli Pseudouridine Synthase RluE

J Mol Biol. 2018 Apr 27;430(9):1284-1294. doi: 10.1016/j.jmb.2018.03.011. Epub 2018 Mar 16.

Abstract

The isomerization of uridine to pseudouridine is the most common type of RNA modification found in RNAs across all domains of life and is performed by RNA-dependent and RNA-independent enzymes. The Escherichia coli pseudouridine synthase RluE acts as a stand-alone, highly specific enzyme forming the universally conserved pseudouridine at position 2457, located in helix 89 (H89) of the 23S rRNA in the peptidyltransferase center. Here, we conduct a detailed structure-function analysis to determine the structural elements both in RluE and in 23S rRNA required for RNA-protein interaction and pseudouridine formation. We determined that RluE recognizes a large part of 23S rRNA comprising both H89 and the single-stranded flanking regions which explains the high substrate specificity of RluE. Within RluE, the target RNA is recognized through sequence-specific contacts with loop L7-8 as well as interactions with loop L1-2 and the flexible N-terminal region. We demonstrate that RluE is a faster pseudouridine synthase than other enzymes which likely enables it to act in the early stages of ribosome formation. In summary, our biochemical characterization of RluE provides detailed insight into the molecular mechanism of RluE forming a highly conserved pseudouridine during ribosome biogenesis.

Keywords: RNA modification; RNA–protein interaction; peptidyltransferase center; pseudouridine; ribosomal RNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Escherichia coli / enzymology*
  • Hydro-Lyases / chemistry*
  • Hydro-Lyases / metabolism*
  • Models, Molecular
  • Nucleic Acid Conformation
  • Protein Binding
  • Protein Conformation
  • Pseudouridine / metabolism
  • RNA, Bacterial / chemistry
  • RNA, Bacterial / genetics
  • RNA, Ribosomal, 23S / chemistry*
  • RNA, Ribosomal, 23S / genetics*
  • Substrate Specificity

Substances

  • RNA, Bacterial
  • RNA, Ribosomal, 23S
  • Pseudouridine
  • Hydro-Lyases
  • pseudouridylate synthetase