MOUSE HEAT-SHOCK TRANSCRIPTION FACTOR-I AND FACTOR-II PREFER A TRIMERIC BINDING-SITE BUT INTERACT DIFFERENTLY WITH THE HSP70 HEAT-SHOCK ELEMENT

To understand the function of multiple heat shock transcription factor s in higher eukaryotes, we have characterized the interaction of recom binant mouse heat shock transcription factors 1 and 2 (mHSF1 and mHSF2 ) with their binding site, the heat shock element (HSE). For our analy sis, we utilized the human HSP70 HSE, which consists of three perfect 5'-nGAAn-3' sites (1, 3, and 4) and two imperfect sites (2 and 5) arra nged as tandem inverted repeats. Recombinant mHSF1 and mHSF2, which ex ist as trimers in solution, both bound specifically to this HSE and st imulated transcription of a human HSP70-CAT construct in vitro. Footpr inting analyses revealed differential binding of mHSF1 and mHSF2 to th e HSP70 HSE. Specifically, mHSF1 bound all five pentameric sites, wher eas mHSF2 failed to interact with the first site of the HSE but bound to sites 2 to 5. Missing-nucleoside analysis demonstrated that the thi rd and fourth nGAAn sites were essential for mHSF1 and mHSF2 binding. The binding of the initial mHSF1 trimer to the HSE exhibited preferenc e for sites 3, 4, and 5, and then binding of a second trimer occurred at sites 1 and 2. These results suggest that HSF may recognize its bin ding site through the dyad symmetry of sites 3 and 4 but requires an a djacent site for stable interaction. Our data demonstrate that mHSF1 a nd mHSF2 bind specifically to the HSE through major groove interaction s. Methidiumpropyl-EDTA footprinting revealed structural differences i n the first and third repeats of the HSE, suggesting that the DNA is d istorted in this region. The possibility that the HSE region is natura lly distorted may assist in understanding how a trimer of HSF can bind to what is essentially an inverted repeat binding site.