Abstract
Most signaling cascades ultimately lead to changes in gene expression by modulating the activity of transcription factors (TFs). The electrophoretic mobility shift assay (EMSA) is a simple but powerful in vitro method for investigation of specific protein–DNA interactions. It makes use of the fact that protein–DNA complexes have a lower electrophoretic mobility in gels than free DNA has. The application of labeled probes in combination with unlabeled competitors allows investigation of DNA-binding specificity and identification of binding motifs with single base-pair resolution. Here we describe the application of EMSAs for the study of interactions of the brassinosteroid-regulated TFs, BRASSINAZOLE-RESISTANT1, (BZR1), BRI1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1)/BZR2, and CESTA with putative binding sites. The classical approach using radiolabeled probes, as well as the more recent application of fluorescent probes, is described and the advantages and disadvantages of both methods are discussed.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Clouse SD (2011) Brassinosteroids. The Arabidopsis Book 9:e0151
Wang Z-Y, Nakano T, Gendron J et al (2002) Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell 2:505–513
Yin Y, Wang Z-Y, Mora-Garcia S et al (2002) BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell 109:181–191
Poppenberger B, Rozhon W, Khan M et al (2011) CESTA, a positive regulator of brassinosteroid biosynthesis. EMBO J 30:1149–1161
Yin Y, Vafeados D, Tao Y et al (2005) A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis. Cell 120:249–259
He J-X, Gendron JM, Sun Y et al (2005) BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science 307:1634–1638 [Erratum Science 308:1743]
Nemhauser JL, Hong F, Chory J (2006) Different plant hormones regulate similar processes through largely nonoverlapping transcriptional responses. Cell 126:467–475
Sun Y, Fan X-Y, Cao D-M et al (2010) Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis. Dev Cell 19:765–777
Hellman LM, Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc 2:1849–1861
Unterholzner SJ, Rozhon W, Papacek M et al (2015) Brassinosteroids are master regulators of gibberellin biosynthesis in Arabidopsis. Plant Cell 27:2261–2272
Fried M, Crothers DM (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res 9:6505–6525
Senear DF, Brenowitz M (1991) Determination of binding constants for cooperative site-specific protein-DNA interactions using the gel mobility-shift assay. J Biol Chem 266:13661–13671
van Oeffelen L, Peeters E, Nguyen Le Minh P et al (2014) The ‘Densitometric Image Analysis Software’ and its application to determine stepwise equilibrium constants from electrophoretic mobility shift assays. PLoS One 9:e85146
Rye HS, Drees BL, Nelson HCM et al (1993) Stable fluorescent dye-DNA complexes in high sensitivity detection of protein-DNA interactions. Application to heat shock transcription factor. J Biol Chem 268:25229–25238
Berger R, Duncan MR, Berman B (1993) Nonradioactive gel mobility shift assay using chemiluminescent detection. Biotechniques 15(650):652
Rodgers JT, Patel P, Hennes JL et al (2000) Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays. Anal Biochem 277:254–259
Ruscher K, Reuter M, Kupper D et al (2000) A fluorescence based non-radioactive electrophoretic mobility shift assay. J Biotechnol 78:163–170
Onizuka T, Endo S, Hirano M et al (2002) Design of a fluorescent electrophoretic mobility shift assay improved for the quantitative and multiple analysis of protein-DNA complexes. Biosci Biotechnol Biochem 66:2732–2734
Rozhon W, Mayerhofer J, Petutschnig E et al (2010) ASKθ, a group-III Arabidopsis GSK3, functions in the brassinosteroid signalling pathway. Plant J 62:215–223
Acknowledgements
The Deutsche Forschungsgemeinschaft (SFB924 TP A12 to B.P.) supported this work. S.J.U. was a member of the TUM graduate school.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Unterholzner, S.J., Rozhon, W., Poppenberger, B. (2017). Analysis of In Vitro DNA Interactions of Brassinosteroid-Controlled Transcription Factors Using Electrophoretic Mobility Shift Assay. In: Russinova, E., Caño-Delgado, A. (eds) Brassinosteroids. Methods in Molecular Biology, vol 1564. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6813-8_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6813-8_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6811-4
Online ISBN: 978-1-4939-6813-8
eBook Packages: Springer Protocols