In vitro activation of purified human heat shock factor by heat.
A major regulatory step in the heat-induced transcription of heat shock protein (hsp) genes in eukaryotes is the activation of heat shock factor (HSF). In metazoans and Schizosaccharomyces 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 binding activity of HSF is a critical component required for activation of heat shock genes. Inactive HSF in extracts of non-heat shocked human cells can be heated in vitro to activate HSF, suggesting the factors required to sense temperature and activate HSF are soluble factors [Larson, J. S., Schuetz, T. J., & Kingston, R. E. (1988) Nature 335, 372-375]. We utilized the ability to purify human HSF in the active form to characterize further the in vitro activation of HSF. Here we have developed a procedure to deactivate the DNA binding ability of HSF. When purified and deactivated HSF is heated, the DNA binding ability of HSF is activated. This activation occurs most efficiently at 43 degrees C (heat shock temperature), but, in contrast to activation in the crude system, some activation of HSF is observed at 37 degrees C (non-heat shock temperature). We show that purified and deactivated HSF is similar to natural inactive HSF in both size and shape. Thus, the monomer to trimer transition that activates HSF can occur in a temperature-dependent fashion in the absence of other proteins. It is possible that these biochemical properties of HSF contribute to the ability of HSF to respond to heat in vivo.
Department of Genetics, Harvard Medical School, Boston, Massachusetts.
J S
JS
Larson
T J
TJ
Schuetz
R E
RE
Kingston
D000818:Animals; D002498:Centrifugation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D011489:Protein Denaturation
D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D004247:DNA