We have cloned two distinct mouse heat shock transcription factor genes, mHSF1 and mHSF2. The mHSF1 and mHSF2 open reading frames are similar in size, containing 503 and 517 amino acids, respectively. Although mHSFI and mHSF2 are quite divergent overall (only 38% identity), they display extensive homology in the DNA-binding and oligomerization domains that are conserved in the heat shock factors of Saccharomyces cerevisiae, Kluyveromyces lactis, Drosophila, tomato, and human. The ability of these two mouse heat shock factors to bind to the heat shock element (HSE) is regulated by heat. mHSFI is expressed in an in vitro translation system in an inactive form that is activated to DNA binding by incubation at temperatures >41~ the same temperatures that activate heat shock factor DNA binding and the stress response in mouse cells in vivo. mHSF2, on the other hand, is expressed in a form that binds DNA constitutively but loses DNA binding by incubation at >41~ Both mHSFI and mHSF2 are encoded by single-copy genes, and neither is transcriptionally regulated by heat shock. However, there is a striking difference in the levels of mHSFI mRNA in different tissues of the mouse.

Drosophila tissue culture cells stimulated by heat shock contain high levels of heat shock activator protein, which binds specifically to the heat-shock control DNA element. In contrast, nonshocked cells have low basal levels of binding activity. Here, we show that within 30 seconds of heat shock of intact cells the sequence-specific binding activity in whole cell extracts increases significantly, reaching a plateau by 5 min after the start of the shock; removal of the heat stimulus returns the activity to basal levels. Known chemical inducers of heat-shock genes elicit a similar pattern of specific binding activity. Moreover, this pattern is observed in the presence of protein synthesis inhibitors, even if the stimulus-withdrawal is repeated sequentially through five cycles. Our results are inconsistent with models which propose proteolysis as the chief means of mediating heat-shock transcriptional control. Rather, they suggest that heat shock activator pre-exists in normal cells in a nonbinding form, which is converted upon cell stimulus to a high affinity, sequence-specific binding form, most probably by a post-translational modification. This conversion may be crucial for the transcriptional activation of heat shock genes.

Drosophila heat shock activator protein, a rare transacting factor which is induced upon heat shock to bind specifically to the heat shock regulatory sequence in vivo, has been purified from shocked cells to more than 95 percent homogeneity by sequence-specific duplex oligonucleotide affinity chromatography. The purified protein has a relative molecular mass of 110 kilodaltons, binds to the regulatory sequence with great affinity and specificity, and strongly stimulates transcription of the Drosophila hsp70 gene. Studies with this regulatory protein should lead to an understanding of the biochemical pathway underlying the heat shock phenomenon.