Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1.
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 acetyl-transferase 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Lawton
M A
MA
Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037.
Dean
S M
SM
Dron
M
M
Kooter
J M
JM
Kragh
K M
KM
Harrison
M J
MJ
Yu
L
L
Tanguay
L
L
Dixon
R A
RA
Lamb
C J
CJ
D000217:Acyltransferases / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002478:Cells, Cultured; D016384:Consensus Sequence; D004247:DNA; D003850:Deoxyribonuclease I; D015252:Deoxyribonucleases, Type II Site-Specific; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D007887:Fabaceae / Q000235:genetics*; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism*; D010946:Plants, Medicinal; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012697:Serine Endopeptidases; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
D010940:Plant Proteins; D014157:Transcription Factors; D004247:DNA; D000217:Acyltransferases; C020141:flavanone synthetase; D003850:Deoxyribonuclease I; C040836:CTAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D012697:Serine Endopeptidases; D019286:Endopeptidase K