DNA GEOMETRY IN TATA BINDING PROTEIN DNA COMPLEXES

描述：TATA结合蛋白(TBP)是DNA结合组分 转录因子IID并识别TATA盒序列 真核基因的启动子区域。TBP的X-射线共晶结构 结合到DNA上的结果表明，这种蛋白质会导致DNA的大弯曲。这个 拟议的研究旨在确定弯道的大小和方向 当TBP与溶液中的DNA结合时，为了将该信息与 晶体结构。方法学以基于DNA环的检测为中心 闭合和高分辨率脚印。释义--解读 实验结果将由蒙特卡罗计算机模拟来辅助 方法：研究方法。建议将观测到的弯曲度进行比较 在晶体和溶液中转录水平不同 启动子将允许评估DNA的功能重要性 弯曲，在TBP结合中，在转录调控中。 这项工作的动机是TBP与DNA结合的X射线结构， 这表明，这种蛋白质会导致很大程度的弯曲和解卷 结合后的DNA中。这种弯曲在转录中很重要 启动，因为弯曲可以改变DNA的外观，从而 通过其他因素(如TFIEB)加强或防止绑定，或弯曲可以 将原本会在DNA上分离的其他蛋白质聚集在一起。 这项研究是关于DNA在溶液中的弯曲和解离 TBP和含TBP的多蛋白质复合体。第一个目标是 TBP在野外诱发弯曲的大小和方向的确定 键入Tata Box和几个变体，使用定量DNA环闭合和 小圆圈装订。含有TATA盒和本征DNA的DNA分子 DNA折弯已经构建完成。当TBP弯曲和固有弯曲时 合作将DNA形成C形，DNA循环非常快， 而当弯曲形成“S”字形时，环化速度很慢。 这种半定量的信息通过蒙特卡洛变得更加定量 卡罗模拟。通过与伯利实验室的合作， 晶体和溶液中观察到的弯曲程度的比较 在突变启动子支持的转录水平下 能够评估DNA弯曲的功能重要性，无论是在 TBP结合的过程和转录的整个过程。 此外，较少有文献记载的AIMS使用类似的技术来研究DNA 弯曲作为其他因素，激活物和阻滞物(例如TFIIB，NC2)结合 TBP-DNA复合体。

DESCRIPTION: TATA-binding protein (TBP) is the DNA-binding component of transcription factor IID and recognizes the TATA box sequence in the promoter region of eukaryotic genes. The x-ray co-crystal structure of TBP bound to DNA shows that the protein induces a large bend in the DNA. The proposed research is aimed at determining the size and direction of the bend when TBP binds to DNA in solution, in order to compare this information with the crystal structure. Methodologies center on assays based on DNA ring closure and high resolution foot printing. The interpretation of experimental results will be aided by Monte Carlo computer simulation methods. It is proposed that comparison of the observed degree of bending in the crystal and in solution with the level of transcription for different promoters will allow the assessment of the functional importance of DNA bending, both in TBP binding, and in the regulation of transcription. The motivation for the work is the X-ray structure of TBP bound to DNA, which shows that the protein induces a large degree of bending and unwinding in the DNA upon binding. This bending is important in transcription initiation, in that the bend can alter the appearance of the DNA and thereby potentiate or prevent binding by other factors (e.g. TFIEB), or bending can bring together other proteins that would otherwise be separated on the DNA. This project concerns the DNA bending and unwinding in solution induced by TBP and TBP-containing multi-protein complexes. The first goal is the determination of the size and direction of the bend induced by TBP at a wild type TATA box and several variants, using quantitative DNA ring closure and minicircle binding. DNA molecules containing a TATA box and an intrinsic DNA bend have been constructed. When the TBP bend and the intrinsic bend cooperate to form the DNA into a C-shape, the DNA cyclizes very rapidly, whereas when the bends form an "S"-shape then cyclization is very slow. This semiqualtitative information is made more quantitative through Monte Carlo simulation. Through a collaboration with the Burley laboratory, comparison of the observed degree of bending in the crystal and in solution with the level of transcription supported by the mutant promoters will enable assessment of the functional importance of DNA bending, both in the process of TBP binding and in the overall process of transcription. Further, less well documented aims utilize similar techniques to study DNA bending as other factors, activators, and repressors (e.g. TFIIB, NC2) bind to the TBP-DNA complex.