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首页> 外文会议>Conference on Single Molecule Spectroscopy and Imaging; 20080119-21; San Jose,CA(US) >Dynamics of TBP binding to the TATA box
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Dynamics of TBP binding to the TATA box

机译：TBP绑定到TATA盒的动力学

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摘要

Gene expression is highly controlled and regulated in living cells. One of the first steps in gene transcription is recognition of the promoter site by the TATA box Binding Protein (TBP). TBP recruits other transcriptions factors and eventually the RNA polymerase II to transcribe the DNA in mRNA. We developed a single pair Forster Resonance Energy Transfer (spFRET) assay to investigate the mechanism of gene regulation. Here, we apply this assay to investigate the initial binding process of TBP to the adenovirus major late (AdML) promoter site. From the spFRET measurements, we were able to identify two conformations of the TBP-DNA complex that correspond to TBP bound in the correct and the opposite orientation. Increased incubation times or the presence of the transcription factor TFHA improved the alignment of TBP on the promoter site. Binding of TBP to the TATA box shows a rich dynamics with abrupt transitions between multiple FRET states. A frame-wise histogram analysis revealed the presence of at least six discrete states, showing that TBP binding is more complicated than previously thought. Hence, the spFRET assay is very sensitive to the conformation of the TBP-DNA complex and is very promising tool for investigating the pathway of TBP binding in detail.
机译：基因表达在活细胞中受到高度控制和调节。基因转录的第一步是通过TATA盒结合蛋白（TBP）识别启动子位点。 TBP会募集其他转录因子，最后募集RNA聚合酶II来转录mRNA中的DNA。我们开发了单对Forster共振能量转移（spFRET）分析方法，以研究基因调控的机制。在这里，我们应用此测定法来研究TBP到腺病毒主要晚期（AdML）启动子位点的初始结合过程。从spFRET测量，我们能够鉴定出TBP-DNA复合物的两个构象，它们对应于以正确和相反方向结合的TBP。延长的孵育时间或转录因子TFHA的存在改善了TBP在启动子位点的比对。将TBP绑定到TATA框显示出丰富的动力学特性，其中多个FRET状态之间突然转换。逐帧直方图分析显示至少存在六个离散状态，这表明TBP绑定比以前认为的要复杂。因此，spFRET分析对TBP-DNA复合物的构象非常敏感，是用于详细研究TBP结合途径的非常有前途的工具。

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