High-Resolution, High-Throughput Analysis of Hfq-Binding Sites Using UV Crosslinking and Analysis of cDNA (CRAC)

  • Brandon Sy
  • Julia Wong
  • Sander Granneman
  • David Tollervey
  • David Gally
  • Jai J. TreeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1737)


Small regulatory nonprotein-coding RNAs (sRNAs) have emerged as ubiquitous and abundant regulators of gene expression in a diverse cross section of bacteria. They play key roles in most aspects of bacterial physiology, including central metabolism, nutrient acquisition, virulence, biofilm formation, and outer membrane composition. RNA sequencing technologies have accelerated the identification of bacterial regulatory RNAs and are now being employed to understand their functions. Many regulatory RNAs require protein partners for activity, or modulate the activity of interacting proteins. Understanding how and where proteins interact with the transcriptome is essential to elucidate the functions of the many sRNAs. Here, we describe the implementation in bacteria of a UV-crosslinking technique termed CRAC that allows stringent, transcriptome-wide recovery of bacterial RNA–protein interaction sites in vivo and at base-pair resolution. We have used CRAC to map protein–RNA interaction sites for the RNA chaperone Hfq and ribonuclease RNase E in pathogenic E. coli, and toxins from toxin–antitoxin systems in Mycobacterium smegmatis, demonstrating the broad applicability of this technique.


Protein–RNA interaction Small RNA Noncoding RNA RBP RNA-binding protein 


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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Brandon Sy
    • 1
  • Julia Wong
    • 1
  • Sander Granneman
    • 2
  • David Tollervey
    • 3
  • David Gally
    • 4
  • Jai J. Tree
    • 1
    Email author
  1. 1.School of Biotechnology and Biomolecular Sciences, University of New South Wales SydneySydneyAustralia
  2. 2.Institute of Structural and Molecular Biology, Centre for Synthetic and Systems Biology (SynthSys), University of EdinburghEdinbughUK
  3. 3.Wellcome Trust Centre for Cell Biology, University of EdinburghEdinburghUK
  4. 4.Division of Infection and ImmunityThe Roslin Institute, University of EdinburghEdinburghUK

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