Mouse heat shock transcription factors 1 and 2 prefer a trimeric binding site but interact differently with the HSP70 heat shock element.
To understand the function of multiple heat shock transcription factors in higher eukaryotes, we have characterized the interaction of recombinant mouse heat shock transcription factors 1 and 2 (mHSF1 and mHSF2) with their binding site, the heat shock element (HSE). For our analysis, 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) arranged as tandem inverted repeats. Recombinant mHSF1 and mHSF2, which exist as trimers in solution, both bound specifically to this HSE and stimulated transcription of a human HSP70-CAT construct in vitro. Footprinting analyses revealed differential binding of mHSF1 and mHSF2 to the HSP70 HSE. Specifically, mHSF1 bound all five pentameric sites, whereas mHSF2 failed to interact with the first site of the HSE but bound to sites 2 to 5. Missing-nucleoside analysis demonstrated that the third and fourth nGAAn sites were essential for mHSF1 and mHSF2 binding. The binding of the initial mHSF1 trimer to the HSE exhibited preference 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 binding site through the dyad symmetry of sites 3 and 4 but requires an adjacent site for stable interaction. Our data demonstrate that mHSF1 and mHSF2 bind specifically to the HSE through major groove interactions. Methidiumpropyl-EDTA footprinting revealed structural differences in the first and third repeats of the HSE, suggesting that the DNA is distorted in this region. The possibility that the HSE region is naturally distorted may assist in understanding how a trimer of HSF can bind to what is essentially an inverted repeat binding site.
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500.
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Kroeger
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Sarge
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Morimoto
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; C070389:HSF2 protein, human; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I