NIRT: Watching Proteins Bend DNA with Subnanometer Resolution

NIRT：以亚纳米分辨率观察蛋白质弯曲 DNA

• 批准号: 0404286
• 负责人: Thomas Perkins
• 金额: $ 149万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0404286&HistoricalAwards=false
• 关键词: NIRT; Watching; Proteins; Bend; DNA

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.

本提案是对NSF 03-043 NIRT类纳米科学与工程倡议的响应。这项跨学科的研究工作将结合高分辨率单分子实验和数学建模与整体生化分析，以研究控制基因表达的蛋白质-DNA相互作用。人类转录因子TATA盒结合蛋白(TBP)是一个更大的蛋白质复合体(TFIID)的中心蛋白质，它与基因中的DNA序列结合，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复合体与DNA结合的机制，以阐明转录启动的关键第一步。将开发一种独特的亚纳米分辨率的光学捕获仪器，以实现生物络合物的单分子研究。改进后的仪器将允许直接检测TBP与TATA-box序列的结合，通过TBP引起的DNA主干100度扭曲导致DNA分子的明显缩短。将仔细协调整体生化实验，以便与创新的单分子结果进行比较。对实验数据的理论分析将确定弯曲角度，并为TBP寻找塔塔盒子所需的搜索时间提供可检验的预测。通过预测蛋白质诱导的DNA弯曲和包裹DNA的不同实验特征，该分析将能够对结合作为DNA张力的函数的结合进行能量描述，并将进一步阐明与DNA结合的多蛋白质复合体的结构。实验和理论分析将检验DNA长度、每个DNA构建的TATA盒序列的数量以及其他DNA结合障碍蛋白的存在如何影响TBP找到TATA盒的时间。该项目采取多方面的教育方法，包括培训本科生和研究生进行研究，在课程开发和科学教学中指导学生，并使用独特的机制向公众解释科学。学生将对生物物理学及其与其他学科的相互关系有深入的了解。跨学科研究团队的联合会议将把具有应用数学、生物和物理背景的学生和研究人员聚集在一起。