Determination of heat-shock transcription factor 2 stoichiometry at looped DNA complexes using scanning force microscopy.
Gene activation frequently requires an array of proteins bound to sites distal to the transcription start site. The assembly of these protein-bound sites into specialized nucleoprotein complexes is a prerequisite for transcriptional activation. Structural analysis of these higher order complexes will provide crucial information for understanding the mechanisms of gene activation. We have used both electron microscopy and scanning force microscopy to elucidate the structure of complexes formed between DNA and heat-shock transcription factor (HSF) 2, a human heat-shock transcriptional activator that binds DNA as a trimer. Electron microscopy reveals that HSF2 will bring together distant DNA sites to create a loop. We show that this association requires only the DNA binding and trimerization domains of HSF2. Metal shadowing techniques used for electron microscopy obscure details of these nucleoprotein structures. Greatly increased resolution was achieved by directly imaging the complexes in the scanning force microscope, which reveals that at least two trimers are required for the association of HSF2-bound DNA sites.
Wyman
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C
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.
Grotkopp
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E
Bustamante
C
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Nelson
H C
HC
D004247:DNA / Q000378:metabolism / Q000648:ultrastructure*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism / Q000648:ultrastructure; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*
D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C070389:HSF2 protein, human; D004247:DNA