Characterization of a nuclear protein that binds to three elements within the silencer region of a bean chalcone synthase gene promoter

M. J. Harrison, M. A. Lawton, C. J. Lamb, R. A. Dixon

Research output: Contribution to journalArticlepeer-review

54 Scopus citations

Abstract

The chalcone synthase (EC 2.3.1.74) gene promoter from the bean Phaseolus vulgaris L. contains a silencer element between positions -140 and -326 from the transcription start site that is functional in electroporated soybean protoplasts. This element contains three binding sites for a bean nuclear factor (SBF-1) with DNA sequence recognition properties that are very similar to those of nuclear factor GT-1. By using a synthetic tetramer of one of the binding sites as probe, we have purified sequence-specific SBF-1 activity approximately 1750-fold from suspension-cell nuclei, by using a combination of ammonium sulfate precipitation, gel filtration, heparin-agarose chromatography, and sequence-specific DNA affinity chromatography. The factor exhibited an apparent molecular weight of 160,000-200,000 on the basis of gel filtration. A subunit molecular weight of approximately 95,000 was determined from SDS/polyacrylamide gel electrophoretic analysis of purified fractions, followed by Southwestern blot analysis (a protein blot probed with oligonucleotide probes), and from UV-cross-linking experiments. The factor lost DNA-binding activity on treatment with alkaline phosphatase. We discuss the properties of SBF-1 in relation to the functionality of GT-1 binding sequences in plant genes.

Original languageEnglish (US)
Pages (from-to)2515-2519
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume88
Issue number6
DOIs
StatePublished - 1991
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Phaseolus vulgaris
  • cis elements
  • trans-acting factor
  • transcription

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