Activation of Drosophila heat shock factor: conformational change associated with a monomer-to-trimer transition.
The induction of heat shock genes in eukaryotic cells is regulated by the transcription factor heat shock factor (HSF). Activation of HSF occurs at two independent levels, DNA binding and the acquisition of transcriptional competence. The binding of HSF to DNA is accomplished by a stress-induced oligomeric switch of HSF protein. We have defined the oligomeric state of the latent and induced forms of HSF by measuring the sedimentation coefficient and the Stokes radius of the protein in Drosophila cell extracts. Calculation of the native molecular mass indicates that the two forms of Drosophila HSF are best described as a monomer and trimer, respectively, of the 77-kDa HSF polypeptide. The monomeric and trimeric states of HSF were verified by chemical cross-linking experiments. The finding of a monomeric composition for the latent form of HSF is incompatible with speculative models which suggest that molecular chaperones such as hsp70 feed back to inhibit trimerization of HSF by forming a stable heteromeric complex. We also found that both HSF monomers and HSF trimers exhibit unusually high frictional ratios, indicating that they have asymmetric shapes. The degree of asymmetry is significantly greater for the HSF trimer, suggesting that the monomer undergoes a conformational change to a more extended structure upon trimerization. These findings are consistent with a model for the inert HSF protein that is based on a monomer constrained by intramolecular coiled-coil interactions between amino- and carboxy-terminal domains.
Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892.
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JT
Westwood
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Wu
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004330:Drosophila / Q000378:metabolism*; D029721:Drosophila Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D000076249:Heat Shock Transcription Factors; D006358:Hot Temperature; D046911:Macromolecular Substances; D008961:Models, Structural; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D014157:Transcription Factors
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D000076249:Heat Shock Transcription Factors; C053134:Hsf protein, Drosophila; D046911:Macromolecular Substances; D010750:Phosphoproteins; D014157:Transcription Factors