Rnase footprinting of protein binding sites on an mRNA target of small RNAs

Yi Peng, Toby J. Soper, Sarah A. Woodson

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Endoribonuclease footprinting is an important technique for probing RNA-protein interactions with single nucleotide resolution. The susceptibility of RNA residues to enzymatic digestion gives information about the RNA secondary structure, the location of protein binding sites, and the effects of protein binding on the RNA structure. Here we present a detailed protocol for using RNase T2, which cleaves single stranded RNA with a preference for A nucleotides, to footprint the protein Hfq on the rpoS mRNA leader. This protocol covers how to form the RNP complex, determine the correct dose of enzyme, footprint the protein, and analyze the cleavage pattern using primer extension.

Original languageEnglish (US)
Title of host publicationBacterial Regulatory RNA
Subtitle of host publicationMethods and Protocols
EditorsKenneth C. Keiler
Pages213-224
Number of pages12
DOIs
StatePublished - Jul 27 2012

Publication series

NameMethods in Molecular Biology
Volume905
ISSN (Print)1064-3745

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Keywords

  • Footprinting
  • Primer extension
  • RNA
  • RNA protein interactions
  • RNase T2
  • Sequencing gel

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Peng, Y., Soper, T. J., & Woodson, S. A. (2012). Rnase footprinting of protein binding sites on an mRNA target of small RNAs. In K. C. Keiler (Ed.), Bacterial Regulatory RNA: Methods and Protocols (pp. 213-224). (Methods in Molecular Biology; Vol. 905). https://doi.org/10.1007/978-1-61779-949-5_13