Partial purification and characterization of an 80-kDa transcription factor binding to bHLH motif in the rat p53 promoter

Minhyung Lee, Yong-beom Lim, Jong Sang Park

Research output: Contribution to journalArticle

Abstract

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°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.

Original languageEnglish
Pages (from-to)337-345
Number of pages9
JournalMolecular Biology Reports
Volume29
Issue number4
DOIs
Publication statusPublished - 2002 Dec 1

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Transcription Factors
p53 Genes
Proteins
Southwestern Blotting
E-Box Elements
Divalent Cations
Affinity Chromatography
Edetic Acid
Oligonucleotides
Urea
Polyacrylamide Gel Electrophoresis
Carrier Proteins
Binding Sites
DNA

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Cite this

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Partial purification and characterization of an 80-kDa transcription factor binding to bHLH motif in the rat p53 promoter. / Lee, Minhyung; Lim, Yong-beom; Park, Jong Sang.

In: Molecular Biology Reports, Vol. 29, No. 4, 01.12.2002, p. 337-345.

Research output: Contribution to journalArticle

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