Detection of three protein binding sites in the serum-regulated promoter of the human gene encoding the 70-kDa heat shock protein

The basal promoter of the human gene encoding 70-kDa heat shock protein (HSP70) controls maximal transcriptional activity and serum-regulated expression. We demonstrate that the three promoter elements defined by in vivo studies - CCAAT, serum-regulated element (SRE), and 'TATA' - correspond to protein-binding sites in vitro. The promoter interactions with protein factors in HeLa cell crude nuclear extracts were detected by an exonuclease III digestion assay. The sequence specificity was demonstrated with promoter probes containing wild-type sequences or unique linker-scanner mutations that alter each of the elements. We suggest that the protein factor binding to the SRE is involved in the serum-regulated expression of the human gene for HSP70.