NIRT: Watching Proteins Bend DNA with Subnanometer Resolution

该提案是响应纳米科学与工程倡议，NSF 03-043，类别NIRT。 这项跨学科的研究工作将结合联合收割机高分辨率的单分子实验和数学建模与合奏生化分析，以研究蛋白质-DNA相互作用，控制基因的表达。人类转录因子TATA盒结合蛋白（TBP）是一个更大的蛋白质复合物（TFIID）的中心蛋白质，结合到基因中的DNA序列，并控制基因如何转录成信使RNA。该项目将研究单个TBP分子和TFIID复合物结合DNA的机制，以阐明转录起始的关键第一步。将开发一种独特的亚纳米分辨率的光学捕获仪器，以实现生物复合物的单分子研究。改进的装置将允许直接检测TBP与TATA盒序列的结合，通过TBP引起的DNA骨架中的100度扭结引起的DNA分子的明显缩短。将仔细协调Ensemble生化实验，以便与创新的单分子结果进行比较。实验数据的理论分析将确定弯曲角，并提供TBP找到TATA盒所需的搜索时间的可测试预测。该分析将使一个充满活力的描述作为DNA张力的函数的结合，并将进一步阐明与DNA结合的多蛋白质复合物的结构，通过预测不同的实验签名的蛋白质诱导的DNA弯曲和包装的DNA周围的复杂。实验和理论分析将研究DNA长度，每个DNA构建体的TATA盒序列数量，以及其他DNA结合障碍蛋白的存在如何影响TBP找到TATA盒的时间。该项目采用多方面的教育方法，包括对本科生和研究生进行研究培训，指导学生进行课程开发和科学教学，用一种独特的机制向公众解释科学。学生将深入了解生物物理学及其与其他学科的相互关系。跨学科研究团队的联席会议将汇集具有应用数学，生物学和物理学背景的学生和研究人员。

This proposal was received in response to Nanoscale Science and Engineering initiative, NSF 03-043, category NIRT. This interdisciplinary research effort will combine high-resolution single-molecule experiments and mathematical modeling with ensemble biochemical assays to study protein-DNA interactions that control gene expression.The human transcription factor TATA box-binding protein (TBP) is the central protein in a larger protein complex (TFIID) that binds to DNA sequences in genes and controls how genes are transcribed into messenger RNA. The proposed project will investigate the mechanism of DNA binding by single TBP molecules and TFIID complexes to illuminate a key first step in transcription initiation. A unique optical trapping instrument with sub-nanometer resolution will be developed to enable single-molecule studies of biological complexes. The improved apparatus will allow direct detection of the binding of TBP to TATA-box sequences, via the apparent shortening of a DNA molecule due to the 100-degree kink in the DNA backbone caused by TBP. Ensemble biochemical experiments will be carefully coordinated for comparison with the innovative single-molecule results. Theoretical analysis of the experimental data will determine the bend angle, and provide testable predictions for the search time required by TBP to find the TATA box. The analysis will enable an energetic description of the binding as a function of DNA tension and will further illuminate the structure of multi-protein complexes bound to DNA, by predicting the different experimental signatures of protein-induced bending of the DNA and wrapping of the DNA around the complex. Experiments and theoretical analysis will examine how DNA length, the number of TATA-box sequences per DNA construct, and the presence of other DNA-binding obstacle proteins affect the time for TBP to find a TATA box.The project takes a multifaceted approach to education that will include training of undergraduate and graduate students in research, mentoring students in course development and science teaching, and using a unique mechanism to explain the science to the general public. Students will develop a deep understanding of biophysics and its interrelation to other disciplines. Joint meetings of the interdisciplinary research team will bring together students and researchers with backgrounds including applied math, biology, and physics.