SILENCER REGION OF A CHALCONE SYNTHASE PROMOTER CONTAINS MULTIPLE BINDING-SITES FOR A FACTOR, SBF-1, CLOSELY RELATED TO GT-1
SILENCER REGION OF A CHALCONE SYNTHASE PROMOTER CONTAINS MULTIPLE BINDING-SITES FOR A FACTOR, SBF-1, CLOSELY RELATED TO GT-1
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DOI：
10.1007/bf00020555
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发表时间：
1991-02-01
影响因子：
5.1
通讯作者：
LAMB, CJ
中科院分区：
文献类型：
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作者：
LAWTON, MA;DEAN, SM;LAMB, CJ
豆核提取物用于凝胶阻滞试验和DNA酶I足迹实验，以确定一种蛋白质因子，命名为SBF-1，它特异性地与豆防御基因CHS 15的启动子中的调控序列相互作用，CHS 15编码类黄酮生物合成酶查耳酮合酶。 SBF-1与-326至-173区域中的三个短序列结合，指定为框1、2和3。 该顺式元件参与植物发育中的器官特异性表达，在来自未分化的悬浮培养大豆细胞的电穿孔原生质体中充当转录沉默子。 沉默元件激活反式共电穿孔的CHS 15-氯霉素乙酰转移酶基因融合体，表明该因子在这些细胞中充当阻遏物。 体外SBF-1结合是快速的、可逆的，并且对先前的热或蛋白酶处理敏感。 竞争性结合试验表明，框1，2和3相互作用的合作，但每个框可以结合因子独立，框3显示最强的结合和框2的最弱的结合。 GGTTAA（A/T）（A/T）（A/T）形成所有三个盒共有的共有序列，类似于豌豆rbcS-3A基因的光响应元件中GT-1因子的结合位点，该基因编码核酮糖二磷酸羧化酶的小亚基。 与CHS 15 - 326至CHS 173元件的结合，以及与盒1、2或3的单独结合，被rbcS-3A的GT-1结合序列竞争，但不被功能失活形式竞争，同样地，CHS序列可以与来自rbcS-3A启动子的真实GT-1位点竞争结合。 这些数据表明SBF-1与GT-1相同或密切相关。
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 CHS 15-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. These data suggest that SBF-1 is identical to, or closely related to, GT-1.