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

• Published in: Proceedings of the National Academy of Sciences - PNAS 1991-03, Vol.88 (6), p.2515-2519
• Main Authors: Harrison, M.J. (The Samuel Roberts Noble Foundation, Ardmore, OK), Lawton, M.A, Lamb, C.J, Dixon, R.A
• Format: Article
• Language: English
• Subjects: Acyltransferases - genetics; Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Cell Nucleus - metabolism; Chromatography; Chromatography, Affinity; Chromatography, Gel; Deoxyribonuclease I; DNA; DNA Probes; EXTRACTOS VEGETALES; EXTRAIT D'ORIGINE VEGETALE; Fabaceae - genetics; Fabaceae - metabolism; Filtration; Fundamental and applied biological sciences. Psychology; Gels; Holoproteins; Immunoblotting; Molecular Sequence Data; Molecular weight; NOYAU CELLULAIRE; Nuclear proteins; Nuclear Proteins - metabolism; NUCLEO; NUCLEOTIDE; NUCLEOTIDOS; PHASEOLUS VULGARIS; Phosphatases; Plant cells; Plants, Medicinal; Promoter regions; Promoter Regions, Genetic; PROTEINAS; PROTEINAS VEGETALES; PROTEINE; PROTEINE VEGETALE; Proteins; Sulfates; TRANSFERASAS; TRANSFERASE
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.88.6.2515

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