DNA GEOMETRY IN TATA BINDING PROTEIN DNA COMPLEXES

TATA 结合蛋白 DNA 复合物中的 DNA 几何结构

• 批准号: 6019117
• 负责人: JASON D KAHN
• 金额: $ 9.48万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019117
• 关键词: DNA binding protein; DNA footprinting; X ray crystallography; computer simulation; genetic promoter element; genetic regulation; genetic transcription; protein structure; transcription factor

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.

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。 TBP的X射线共晶结构 与DNA结合的结果表明，该蛋白质诱导DNA中的大弯曲。 的 建议的研究旨在确定弯曲的尺寸和方向 当TBP与溶液中的DNA结合时，为了将此信息与 晶体结构。 方法学以基于DNA环的分析为中心 封闭和高分辨率的足迹印刷。 的解释 实验结果将得到蒙特卡罗计算机模拟的辅助 方法. 建议将观测到的弯曲程度 在晶体中和溶液中的转录水平不同， 启动子将允许评估DNA的功能重要性， 弯曲，无论是在TBP结合还是在转录调节中。 这项工作的动机是TBP与DNA结合的X射线结构， 这表明蛋白质诱导了很大程度的弯曲和解旋 在DNA中结合。 这种弯曲在转录中很重要 起始，因为弯曲可以改变DNA的外观，从而 增强或防止其他因素（例如TFIEB）的结合，或弯曲可 将原本在DNA上分离的其他蛋白质聚集在一起。 本项目研究了DNA在溶液中的弯曲和解旋， TBP和含TBP的多蛋白复合物。 第一个目标是 在野外测定TBP引起的弯曲的大小和方向 型TATA盒和几种变体，使用定量DNA环闭合， 微环结合 含有TATA盒和内在的 构建了DNA弯曲。 当TBP弯曲和固有弯曲 合作形成一个C形的DNA，DNA环化非常迅速， 而当弯曲形成“S”形时，则环化非常慢。 这种半定性的信息是通过蒙特 Carlo模拟 通过与白肋烟实验室的合作， 晶体和溶液中观察到的弯曲程度的比较 突变启动子支持的转录水平将 能够评估DNA弯曲的功能重要性， TBP结合过程和整个转录过程。 此外，文献较少的目标利用类似的技术来研究DNA 当其他因子、激活因子和抑制因子（例如TFIIB、NC 2）结合时， TBP-DNA复合物。