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 site s distal to the transcription start site. The assembly of these protei n-bound sites into specialized nucleoprotein complexes is a prerequisi te for transcriptional activation. Structural analysis of these higher order complexes will provide crucial information for understanding th e 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 hum an heat-shock transcriptional activator that binds DNA as a trimer. El ectron microscopy reveals that HSF2 will bring together distant DNA si tes to create a loop. We show that this association requires only the DNA binding and trimerization domains of HSF2. Metal shadowing techniq ues used for electron microscopy obscure details of these nucleoprotei n structures. Greatly increased resolution was achieved by directly im aging the complexes in the scanning force microscope, which reveals th at at least two trimers are required for the association of HSF2-bound DNA sites.