Sequence-Specific Affinity Chromatography of Bacterial Small Regulatory RNA-Binding Proteins from Bacterial Cells

Methods Mol Biol. 2018:1737:341-350. doi: 10.1007/978-1-4939-7634-8_19.

Abstract

Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules.

Keywords: Affinity chromatography; Bacterial small RNAs; Hfq; In vivo crosslinking; RNA-binding proteins; Western blot.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Chromatography, Affinity / methods*
  • Cross-Linking Reagents / metabolism
  • Protein Binding
  • Pseudomonas aeruginosa / genetics
  • Pseudomonas aeruginosa / growth & development
  • Pseudomonas aeruginosa / metabolism*
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism*
  • RNA, Small Untranslated / genetics
  • RNA, Small Untranslated / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / isolation & purification
  • RNA-Binding Proteins / metabolism*
  • Ultraviolet Rays

Substances

  • Bacterial Proteins
  • Cross-Linking Reagents
  • RNA, Bacterial
  • RNA, Small Untranslated
  • RNA-Binding Proteins