COMPLEX MECHANISM OF TBP BINDING TO DNA

我们的长期目标是在分子水平上了解真核生物中基因转录的启动和调控机制。第一步是确定TATA结合蛋白(TBP)与特定DNA序列结合的机制。转录起始的第一步是将TBP与转录起始上游的启动子元件25碱基对结合。TFIIB蛋白接下来结合形成三元复合体。现在可以得到几个二元TBP-DNA络合物以及三元络合物：TBP：DNA：TFIIB的晶体结构。随后，其他蛋白质被招募，直到启动前的TATA盒。TBP与DNA的小凹槽结合，使凹槽变宽，并在TATA盒的第一步和最后一步产生两个尖锐的扭结，在每个位置插入一对苯丙烷。DNA在盒子内解开，尽管DNA总体上有80度的结合，但沃森-克里克的碱基配对保持不变。DNA的这种不寻常的结构变化与复杂的结合动力学有关。我们理解这一机制的途径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的弯曲和结合。该方法包括改变启动子序列、TBP和溶液条件，以便绘制出反应路径上的能量和结构变化。实验将检验这一假设，即TBP+DNA反应中的中间产物在确保产生的启动子序列导致正确而快速的PIC组装方面发挥着重要作用。从总体上看，很明显，基因转录调控在疾病和健康状态下都是重要的，了解调控的机制细节可以为治疗设计提供一种合理的方法。许多疾病，包括许多癌症、哮喘、心脏病，也许还有阿尔茨海默病，都与蛋白质合成的改变直接相关，在许多情况下，转录的改变已经被证明。

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.