Abstract
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.
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References
Vogel J, Luisi BF (2011) Hfq and its constellation of RNA. Nat Rev Microbiol 9:578–589
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 10:636–651
Kaberdin VR, Blasi U (2006) Translation initiation and the fate of bacterial mRNAs. FEMS Microbiol Rev 30:967–979
Gottesman S, McCullen CA, Guillier M et al (2006) Small RNA regulators and the bacterial response to stress. Cold Spring Harb Symp Quant Biol 71:1–11
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 27:552–564
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 21:3546–3556
Link TM, Valentin-Hansen P, Brennan RG (2009) Structure of Escherichia coli Hfq bound to polyriboadenylate RNA. Proc Natl Acad Sci U S A 106:19292–19297
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 11:1206–1214
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 109:9396–9401
Panja S, Schu DJ, Woodson SA (2013) Conserved arginines on the rim of Hfq catalyze base pair formation and exchange. Nucleic Acids Res 41:7536–7546
Ali Azam T, Iwata A, Nishimura A et al (1999) Growth phase-dependent variation in protein composition of the Escherichia coli nucleoid. J Bacteriol 181:6361–6370
Wu P, Brand L (1994) Resonance energy transfer: methods and applications. Anal Biochem 218:1–13
Hopkins JF, Panja S, Woodson SA (2011) Rapid binding and release of Hfq from ternary complexes during RNA annealing. Nucleic Acids Res 39:5193–5202
Kim H, Abeysirigunawardena SC, Chen K et al (2014) Protein-guided RNA dynamics during early ribosome assembly. Nature 506:334–338
Wittig I, Schägger H (2005) Advantages and limitations of clear-native PAGE. Proteomics 5:4338–4346
Updegrove TB, Correia JJ, Chen Y et al (2011) The stoichiometry of the Escherichia coli Hfq protein bound to RNA. RNA 17:489–500
Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, Baltimore, Chapter 10–12
Chen RF (1968) Dansyl labeled proteins: determination of extinction coefficient and number of bound residues with radioactive dansyl chloride. Anal Biochem 25:412–416
Panja S, Woodson SA (2012) Hexamer to monomer equilibrium of E. coli Hfq in solution and its impact on RNA annealing. J Mol Biol 417:406–412
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 35:999–1006
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 41:487–497
Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308
Rajkowitsch L, Schroeder R (2007) Dissecting RNA chaperone activity. RNA 13:2053–2060
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 37:6205–6213
Peng Y, Soper TJ, Woodson SA (2014) Positional effects of AAN motifs in rpoS regulation by sRNAs and Hfq. J Mol Biol 426:275–285
Hopkins JF, Woodson SA (2005) Molecular beacons as probes of RNA unfolding under native conditions. Nucleic Acids Res 33:5763–5770
Acknowledgements
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.
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Panja, S., Woodson, S.A. (2015). Fluorescence Reporters for Hfq Oligomerization and RNA Annealing. In: Boudvillain, M. (eds) RNA Remodeling Proteins. Methods in Molecular Biology, vol 1259. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2214-7_22
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DOI: https://doi.org/10.1007/978-1-4939-2214-7_22
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