COMPLEX MECHANISM OF TBP BINDING TO DNA

TBP 与 DNA 结合的复杂机制

• 批准号: 6525522
• 负责人: Lawrence JOHN Parkhurst
• 金额: $ 19.71万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525522
• 关键词: DNA; DNA binding protein; chemical kinetics; conformation; fluorescence resonance energy transfer; fluorescent dye /probe; genetic promoter element; genetic transcription; intermolecular interaction; ionic strengths; nucleic acid sequence; osmotic pressure; protein binding; stop flow technique; thermodynamics; transcription factor

Our long-term goals is to understand the mechanism by which gene transcription in eukaryotes is initiated and regulated at the molecular level. The first step is to determine the mechanism by which the TATA binding protein (TBP) binds to specific sequences of DNA. The first step in the initiation of transcription is the binding of TBP to a promoter element 25 base pairs upstream from the initiation of transcription. The protein TFIIB binds next to form a ternary complex. Crystal structures are now available for several binary TBP-DNA complexes as well as for the ternary complex: TBP:DNA:TFIIB. Subsequently, other proteins are recruited until the pre- initiation TATA box. TBP binds to the minor groove of the DNA, widening the groove and producing two sharp kinks at the first and last steps of the TATA box where at each site a pair of phenylalannes is intercalated. The DNA is unwound within the box, and despite the overall 80 degree bind in the DNA, Watson-Crick base pairing is maintained. This unusual structural change in the DNA is associated with complex binding kinetics. Our approach to understanding the mechanism is to use rapid reaction kinetic techniques, combined with equilibrium measurements and a fluorescence detection method that can monitor the DNA bending and binding in real time. The approach involves varying the promoter sequence, the TBP, and solution conditions in order to map out the energetics and structural change along the reaction path. Experiments will test the hypothesis that intermediates in the TBP+DNA reaction play important roles in assuring that productive promoter sequences lead to proper and rapid assembly of the PIC. From an overall perspective it is clear that the regulation of gene transcription must be important in states of disease and of health and that understanding the mechanistic details of the regulation affords a rational approach to therapeutic design. A number of diseases, including many cancers, asthma, heart disease, and perhaps Alzheimer's disease are directly linked to altered synthesis of proteins and in a number of cases alterations in transcription have been demonstrated.

我们的长期目标是了解在分子水平启动和调节真核生物中基因转录的机制。第一步是确定TATA结合蛋白（TBP）与DNA的特定序列结合的机制。转录开始的第一步是TBP与转录开始上游上游的启动子元素对25碱基对的结合。蛋白质TFIIB结合在形成三元络合物旁边。现在可以适用于几种二元TBP-DNA复合物以及三元复合物：TBP：DNA：TFIIB。随后，招募了其他蛋白质，直到启动tata盒为止。 TBP与DNA的次要凹槽结合，在塔塔盒的第一和最后一步中拓宽了凹槽，并产生了两个尖锐的扭结，在每个位置，在每个地点，一对苯丙烷被插入。 DNA在盒子内开明，尽管DNA中的总体80度结合，但仍保持了沃森 - 克里克基底配对。 DNA中这种异常的结构变化与复杂的结合动力学有关。我们理解该机制的方法是使用快速反应动力学技术，结合平衡测量和荧光检测方法，可以实时监测DNA弯曲和结合。该方法涉及改变启动子序列，TBP和溶液条件，以绘制沿反应路径的能量和结构变化。实验将测试以下假设：TBP+DNA反应中的中间体在确保生产启动子序列导致PIC的适当和快速组装方面起着重要作用。从总体角度来看，很明显，基因转录的调节在疾病状态和健康状态中必须很重要，并且了解调节的机理细节为治疗设计提供了合理的方法。许多疾病，包括许多癌症，哮喘，心脏病，也许是阿尔茨海默氏病直接与蛋白质的合成改变有关，并且在许多情况下已证明了转录的改变。