Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1

Michael A. Lawton, Susan M. Dean, Michel Dron, Jan M. Kooter, Karsten M. Kragh, Maria J. Harrison, Lloyd Yu, Lisa Tanguay, Richard A. Dixon, Christopher J. Lamb

Research output: Contribution to journalArticlepeer-review

97 Scopus citations

Abstract

Bean nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor, designated SBF-1, that specifically interacts with regulatory sequences in the promoter of the bean defense gene CHS15, which encodes the flavonoid biosynthetic enzyme chalcone synthase. SBF-1 binds to three short sequences designated boxes 1, 2 and 3 in the region -326 to -173. This cis-element, which is involved in organ-specific expression in plant development, functions as a transcriptional silencer in electroporated protoplasts derived from undifferentiated suspension-cultured soybean cells. The silencer element activates in trans a co-electroporated CHS15-chloramphenicol acetyltransferase gene fusion, indicating that the factor acts as a repressor in these cells. SBF-1 binding in vitro is rapid, reversible and sensitive to prior heat or protease treatment. Competitive binding assays show that boxes 1, 2 and 3 interact cooperatively, but that each box can bind the factor independently, with box 3 showing the strongest binding and box 2 the weakest binding. GGTTAA(A/T)(A/T)(A/T), which forms a consensus sequence common to all three boxes, resembles the binding site for the GT-1 factor in light-responsive elements of the pea rbcS-3A gene, which encodes the small subunit of ribulose bisphosphate carboxylase. Binding to the CHS15 -326 to -173 element, and to boxes 1, 2 or 3 individually, is competed by the GT-1 binding sequence of rbcS-3A, but not by a functionally inactive form, and likewise the CHS sequences can compete with authentic GT-1 sites from the rbcS-3A promoter for binding. These data suggest that SBF-1 is identical to, or closely related to, GT-1.

Original languageEnglish (US)
Pages (from-to)235-249
Number of pages15
JournalPlant Molecular Biology
Volume16
Issue number2
DOIs
StatePublished - Feb 1991
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Agronomy and Crop Science
  • Genetics
  • Plant Science

Keywords

  • GT-1
  • chalcone synthase
  • cis-elements
  • silencer
  • transcription

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