Fluorescence Reporters for Hfq Oligomerization and RNA Annealing

  • Subrata Panja
  • Sarah A. WoodsonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)


Fluorescence spectroscopy is a sensitive technique for detecting protein–protein, protein–RNA, and RNA–RNA interactions, requiring only nanomolar concentrations of labeled components. Fluorescence anisotropy provides information about the assembly of multi-subunit proteins, while molecular beacons provide a sensitive and quantitative reporter for base pairing between complementary RNAs. Here we present a detailed protocol for labeling Hfq protein with cyanine 3-maleimide and dansyl chloride to study the protein oligomerization and RNA binding by semi-native polyacrylamide gel electrophoresis (PAGE) and fluorescence anisotropy. We also present a detailed protocol for measuring the rate of annealing between a molecular beacon and a target RNA in the presence of Hfq using a stopped-flow spectrometer.

Key words

Protein oligomerization RNA chaperone Molecular beacon Fluorescence anisotropy Stopped-flow fluorescence Hfq 



The authors thank T. Soper, Y. Peng, and A. Santiago-Frangos for helpful discussion. This work was supported by a grant from the NIH R01 GM46686.


  1. 1.
    Vogel J, Luisi BF (2011) Hfq and its constellation of RNA. Nat Rev Microbiol 9:578–589PubMedCrossRefGoogle Scholar
  2. 2.
    Mura C, Randolph PS, Patterson J, Cozen AE (2013) Archaeal and eukaryotic homologs of Hfq: a structural and evolutionary perspective on Sm function. RNA Biol 10:636–651PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Kaberdin VR, Blasi U (2006) Translation initiation and the fate of bacterial mRNAs. FEMS Microbiol Rev 30:967–979PubMedCrossRefGoogle Scholar
  4. 4.
    Gottesman S, McCullen CA, Guillier M et al (2006) Small RNA regulators and the bacterial response to stress. Cold Spring Harb Symp Quant Biol 71:1–11PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Göpel Y, Papenfort K, Reichenbach B et al (2013) Targeted decay of a regulatory small RNA by an adaptor protein for RNase E and counteraction by an anti-adaptor RNA. Genes Dev 27:552–564PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Schumacher MA, Pearson RF, Moller T et al (2002) Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein. EMBO J 21:3546–3556PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Link TM, Valentin-Hansen P, Brennan RG (2009) Structure of Escherichia coli Hfq bound to polyriboadenylate RNA. Proc Natl Acad Sci U S A 106:19292–19297PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Mikulecky PJ, Kaw MK, Brescia CC et al (2004) Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs. Nat Struct Mol Biol 11:1206–1214PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Sauer E, Schmidt S, Weichenrieder O (2012) Small RNA binding to the lateral surface of Hfq hexamers and structural rearrangements upon mRNA target recognition. Proc Natl Acad Sci U S A 109:9396–9401PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Panja S, Schu DJ, Woodson SA (2013) Conserved arginines on the rim of Hfq catalyze base pair formation and exchange. Nucleic Acids Res 41:7536–7546PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Ali Azam T, Iwata A, Nishimura A et al (1999) Growth phase-dependent variation in protein composition of the Escherichia coli nucleoid. J Bacteriol 181:6361–6370PubMedCentralPubMedGoogle Scholar
  12. 12.
    Wu P, Brand L (1994) Resonance energy transfer: methods and applications. Anal Biochem 218:1–13PubMedCrossRefGoogle Scholar
  13. 13.
    Hopkins JF, Panja S, Woodson SA (2011) Rapid binding and release of Hfq from ternary complexes during RNA annealing. Nucleic Acids Res 39:5193–5202PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Kim H, Abeysirigunawardena SC, Chen K et al (2014) Protein-guided RNA dynamics during early ribosome assembly. Nature 506:334–338PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Wittig I, Schägger H (2005) Advantages and limitations of clear-native PAGE. Proteomics 5:4338–4346PubMedCrossRefGoogle Scholar
  16. 16.
    Updegrove TB, Correia JJ, Chen Y et al (2011) The stoichiometry of the Escherichia coli Hfq protein bound to RNA. RNA 17:489–500PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, Baltimore, Chapter 10–12CrossRefGoogle Scholar
  18. 18.
    Chen RF (1968) Dansyl labeled proteins: determination of extinction coefficient and number of bound residues with radioactive dansyl chloride. Anal Biochem 25:412–416PubMedCrossRefGoogle Scholar
  19. 19.
    Panja S, Woodson SA (2012) Hexamer to monomer equilibrium of E. coli Hfq in solution and its impact on RNA annealing. J Mol Biol 417:406–412PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Arluison V, Hohng S, Roy R et al (2007) Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA. Nucleic Acids Res 35:999–1006PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Doetsch M, Stampfl S, Fürtig B et al (2013) Study of E. coli Hfq's RNA annealing acceleration and duplex destabilization activities using substrates with different GC-contents. Nucleic Acids Res 41:487–497PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308PubMedCrossRefGoogle Scholar
  23. 23.
    Rajkowitsch L, Schroeder R (2007) Dissecting RNA chaperone activity. RNA 13:2053–2060PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Hopkins JF, Panja S, McNeil SA, Woodson SA (2009) Effect of salt and RNA structure on annealing and strand displacement by Hfq. Nucleic Acids Res 37:6205–6213PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Peng Y, Soper TJ, Woodson SA (2014) Positional effects of AAN motifs in rpoS regulation by sRNAs and Hfq. J Mol Biol 426:275–285PubMedCrossRefGoogle Scholar
  26. 26.
    Hopkins JF, Woodson SA (2005) Molecular beacons as probes of RNA unfolding under native conditions. Nucleic Acids Res 33:5763–5770PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.T. C. Jenkins Department of BiophysicsJohns Hopkins UniversityBaltimoreUSA

Personalised recommendations