Receptor-DNA interactions: EMSA and footprinting.
Defining the precise promoter DNA sequence motifs where nuclear receptors and other transcription factors bind is an essential prerequisite for understanding how these proteins modulate the expression of their specific target genes. The purpose of this chapter is to provide the reader with a detailed guide with respect to the materials and the key methods required to perform this type of DNA-binding analysis. Irrespective of whether starting with purified DNA-binding proteins or somewhat crude cellular extracts, the tried-and-true procedures described here will enable one to accurately access the capacity of specific proteins to bind to DNA as well as to determine the exact sequences and DNA contact nucleotides involved. For illustrative purposes, we primarily have used the interaction of the androgen receptor with the rat probasin proximal promoter as our model system.
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Jason T
JT
Department of Urologic Sciences, Prostate Center, Vancouver General Hospital, University of British Columbia, Vancouver, BC, Canada.
Cheng
Helen
H
Hendy
Stephen C
SC
Nelson
Colleen C
CC
Rennie
Paul S
PS
D000727:Androgen-Binding Protein / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D018983:DNA Footprinting / Q000295:instrumentation / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000295:instrumentation / Q000379:methods*; D008745:Methylation; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism
D000727:Androgen-Binding Protein; D011944:Receptors, Androgen; C027519:probasin