Identification of a transcription factor, an 80-kDa protein that interactswith the HLH recognition motif of the rat p53 promoter

The p53 promoter has been shown to contain a number of potential regulatory motifs. It was previously reported that the upstream stimulating factor (U SF) played a central role in regulating the p53 expression. The USF binding site, E-box, is located around 40 bp upstream of the major transcription s tart site. In this study, it was confirmed that the E-box binds to proteins by DNase I footprinting assay. In the electrophoretic mobility shift assay (EMSA), two retarded bands were detected. One band was abolished by the co mpetition of USF consensus oligonucleotide, but the other band was not. Thi s result indicated that a factor, other than USF, was bound to the E-box. T he molecular masses of the binding proteins were determined by a Southweste rn-blotting assay. As a result, 46- and 80-kDa proteins were detected. The 46-kDa protein was eliminated by the competition of USF consensus oligonucl eotide. Also, the Southwestern-blotting assay with P-32-labeled USF consens us oligonucleotide showed only a 46-kDa protein. Therefore, the 46-kDa prot ein was USF. These results showed that USF and the 80-kDa protein were boun d to the E-box. In addition, it was proved by in vitro transcription assay that this 80-kDa protein had a basal transcriptional activity.