MEASUREMENT OF THE BINDING OF TRANSCRIPTION FACTOR SP1 TO A SINGLE GC BOX RECOGNITION SEQUENCE
MEASUREMENT OF THE BINDING OF TRANSCRIPTION FACTOR SP1 TO A SINGLE GC BOX RECOGNITION SEQUENCE
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DOI：
10.1093/nar/17.7.2639
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发表时间：
1989-04-11
影响因子：
14.9
通讯作者：
DYNAN, WS
中科院分区：
文献类型：
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作者：
LETOVSKY, J;DYNAN, WS
测定转录因子Sp1与单一一致性CG盒DNA识别位点（5′-GGGGCGGGGC-3′）结合的平衡常数。在这些实验中，识别位点的单个拷贝被合成并在标准质粒背景下克隆。结合可以通过足迹测定法来测量，从而使结合反应处于平衡状态，或者通过凝胶迁移转移测定法来测量。通过计算机辅助拟合理论曲线，确定了反应中活性Sp1的浓度和解离常数。不同实验得到的解离常数值为4.1倍。10-10米至5.3倍。10-10 M.还测试了共识识别位点的几个变体。a -取代突变体（5”- GGGG_AGGGGC-3”）和t -取代突变体（5”-GGGG_TGGGGC-3”）的结合强度分别比共识位点弱3倍和6倍。一个g取代变体（5' -GGGG_GGGGGC-3'）的结合强度至少比共识位点弱30倍。这些发现有助于区分Sp1-DNA识别的不同模型。它们与Sp1和中心C-G碱基对之间存在特定的氢键接触一致，但没有提供特别的证据来支持局部DNA结构是相互作用的主要因素的模型。
The equilibrium constant was determined for the binding of the transcription factor Sp1 to a single consensus CG box DNA recognition site, (5''-GGGGCGGGGC-3''). For these experiments, single copies of the recognition site were synthesized and cloned in a standard plasmid background. Binding was measured either by a footprinting assay modified so that the binding reaction was at equilibrium, or by a gel mobility shift assay. The concentration of active Sp1 in the reactions and the dissociation constant were determined by computer-assisted fitting to theoretical curves. Values for the dissociation constant obtained in different experiments ranged from 4.1 .times. 10-10 M to 5.3 .times. 10-10 M. Several variants of the consensus recognition site were also tested. An A-substituted variant (5''GGGG_AGGGGC-3'') and a T-substituted variant (5''-GGGG_TGGGGC-3'') were bound 3-fold and 6-fold more weakly than the consensus site, respectively. A G-substituted variant (5''-GGGG_GGGGGC-3'') was bound at least 30-fold more weakly than the consensus site. These findings help distinguish between alternative models for Sp1-DNA recognition. They are consistent with the presence of specific hydrogen-bond contacts between Sp1 and the central C-G base pair, but provide no particular evidence to support a model where local DNA structure is the dominant factor in the interaction.