IN-VITRO ACTIVATION OF PURIFIED HUMAN HEAT-SHOCK FACTOR BY HEAT

A major regulatory step in the heat-induced transcription of heat shoc k protein (hsp) genes in eukaryotes is the activation of heat shock fa ctor (HSF). In metazoans and Schizasaccharomyces pombe, HSF is present in unstressed cells but is unable to bind to its target DNA sequence element, the heat shock element (HSE). Heat induction of the DNA bindi ng activity of HSF is a critical component required for activation of heat shock genes. Inactive HSF in extracts of non-heat shocked human c ells can be heated in vitro to activate HSF, suggesting the factors re quired to sense temperature and activate HSF are soluble factors [Lars on, J. S., Schuetz, T. J., and Kingston, R. E. (1988) Nature 335, 372- 375]. We utilized the ability to purify human HSF in the active form t o characterize further the in vitro activation of HSF. Here we have de veloped a procedure to deactivate the DNA binding ability of HSF. When purified and deactivated HSF is heated, the DNA binding ability of HS F is activated. This activation occurs most efficiently at 43 degrees C (heat shock temperature), but, in contrast to activation in the crud e system, some activation of HSF is observed at 37 degrees C (non-heat shock temperature). We show that purified and deactivated HSF is simi lar to natural inactive HSF in both size and shape. Thus, the monomer to trimer transition that activates HSF can occur in a temperature-dep endent fashion in the absence of other proteins. It is possible that t hese biochemical properties of HSF contribute to the ability of HSF to respond to heat in vivo.