Partial purification and characterization of an 80-kDa transcription factor binding to bHLH motif in the rat p53 promoter.
E-box is one of potential cis-regulatory elements for the p53 gene. It was previously reported that USF bound to the E-box of the p53 gene. Recently, we demonstrated that an 80-kDa protein other than USF bound to the E-box and activated the transcription of the p53 gene. In the present study, the 80-kDa protein was partially purified and characterized. First, we confirmed that nuclear factors bound to the E-box in sequence-specific manner by the oligonucleotide competition assay. The binding protein to the E-box was partially purified by a sequence-specific DNA affinity chromatography. The active fraction was analyzed by SDS-PAGE and southwestern blotting assay, which showed that the 80-kDa protein was enriched. The binding activity of the 80-kDa protein was not decreased in the presence of 1.4 M urea. In addition, the binding activity was stable up to 50 degrees C. Treatment of EDTA showed that the 80-kDa protein did not require divalent cation such as Mg2+ for the maximum DNA binding activity. The competition assay with non-specific competitor, poly (dI-dC) showed that the 80-kDa protein had high affinity to its binding site. These biochemical properties provide useful insights into the 80-kDa nuclear factor binding to the p53 promoter.
Lee
Minhyung
M
School of Chemistry & Molecular Engineering, Seoul National University, Seoul 151-742, Korea.
Lim
Yong-beom
YB
Park
Jong-sang
JS
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D004268:DNA-Binding Proteins; D004492:Edetic Acid / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D018257:Helix-Loop-Helix Motifs; D006358:Hot Temperature; D008970:Molecular Weight; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D020218:Response Elements / Q000235:genetics*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D051720:Upstream Stimulatory Factors; D014508:Urea / Q000494:pharmacology
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; D014508:Urea; D004492:Edetic Acid