NMR Studies of the DNA Target Search Process by a Multi-zinc-Finger Protein

多锌指蛋白 DNA 靶标搜索过程的 NMR 研究

• 批准号: 0920238
• 负责人: Junji Iwahara
• 金额: $ 40.8万
• 依托单位: University of Texas Medical Branch at Galveston
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0920238&HistoricalAwards=false
• 关键词: NMR; Studies; DNA; Target; Search

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Gene-regulation by DNA-binding proteins is vital for living systems. Such proteins control gene expression by binding to particular DNA sequences, and the association must be rapid for quick response to cellular signals. The overall objective of this project is to obtain a physical understanding of the target search process whereby DNA-binding proteins rapidly and efficiently locate their target DNA sites in the presence of an enormous background of nonspecific DNA. In particular, the research component of this three-year project focuses on the structural, dynamic, and kinetic aspects of the target search by a gene-regulatory protein with multiple zinc-finger DNA-binding domains (DBDs). Using NMR spectroscopy, this project will analyze the target search by the Zif268 protein that contains three zinc-finger DBDs as a model system. This protein is an inducible gene-regulatory protein involved in neuronal plasticity and memory formation, and must efficiently find its target DNA sequences to mediate rapid cellular responses to neuronal signals. The Zif268 protein is an ideal model for the study of the target search process, because it has been extensively characterized in biochemical, structural and functional terms, and also exhibits excellent NMR spectra for both specific and non-specific DNA complexes. This project will address three major questions: 1) In terms of structure and dynamics, how does the Zif268 protein use the three DBDs for the target search while it moves on the nonspecific DNA 2) How efficient is the so-called direct transfer mechanism for the Zif268 protein 3) What is the relative contribution of the sliding mechanism to the target search at high DNA concentrations To answer these questions, various NMR approaches that have been developed in their previous studies will be used. This project will provide important and general insights into the target search by zinc-finger proteins, the most abundant class of eukaryotic gene-regulatory proteins. Experimental data obtained in this project will immediately be shared and discussed with Dr. Yaakov Levy's group (Weizmann Institute of Science, Israel), which has been studying theoretical and computational aspects of the facilitated target location by DNA-binding proteins. This international collaboration will integrate experimental and theoretical approaches to lead to more realistic views of the target search process. Broader Impacts: By integrating research and education, this project aims to establish an institutional environment where students and researchers can effectively take advantage of NMR spectroscopy in their research. The education component offers opportunities whereby graduate students and research staff in the University of Texas Medical Branch (UTMB) can learn NMR as a research tool. The principal investigator (PI) of this project will design Mathematica-based syllabus for NMR spectroscopy, and also initiate an elective course to teach practical aspects of NMR spectroscopy, demonstrating actual data collection and analysis. Furthermore, the PI will continue to organize the NMR seminar as an effective means to share knowledge and tools. The educational component of this project includes science education for high school students and international student exchange with the collaborators laboratory. Through UTMB's HSSRP program, high school students are involved in the research in the context of this project. This offers a fascinating project for high school students, in which they combine their separate knowledge in biology, chemistry, physics and mathematics to conduct scientific research in a professional environment. The high school students will be supervised primarily by the graduate students involved in this project, which will give the UTMB students an excellent opportunity to teach at an institution with no regular undergraduate program. The international student exchange will provide opportunities for graduate students with different cultural backgrounds to learn the importance of wider vision in science, because each of the two groups is investigating the same subject by totally different and complementary approaches.

该奖项由 2009 年美国复苏和再投资法案（公法 111-5）资助。DNA 结合蛋白的基因调控对于生命系统至关重要。此类蛋白质通过与特定 DNA 序列结合来控制基因表达，并且这种结合必须快速，以便对细胞信号做出快速反应。该项目的总体目标是获得对目标搜索过程的物理理解，通过该过程，DNA 结合蛋白在大量非特异性 DNA 背景下快速有效地定位其目标 DNA 位点。特别是，这个为期三年的项目的研究部分重点关注具有多个锌指 DNA 结合域 (DBD) 的基因调节蛋白进行靶标搜索的结构、动态和动力学方面。该项目将使用核磁共振波谱分析 Zif268 蛋白的靶标搜索，该蛋白包含三个锌指 DBD 作为模型系统。该蛋白是一种诱导型基因调节蛋白，参与神经元可塑性和记忆形成，必须有效地找到其目标 DNA 序列以介导细胞对神经元信号的快速反应。 Zif268 蛋白是研究靶标搜索过程的理想模型，因为它在生化、结构和功能方面得到了广泛的表征，并且还对特定和非特定 DNA 复合物表现出出色的 NMR 谱。该项目将解决三个主要问题：1）在结构和动力学方面，Zif268蛋白在非特异性DNA上移动时如何利用三个DBD进行目标搜索2）Zif268蛋白所谓的直接转移机制的效率如何3）滑动机制在高DNA浓度下对目标搜索的相对贡献是什么为了回答这些问题，他们在之前的研究中开发了各种NMR方法 将被使用。该项目将为锌指蛋白（最丰富的一类真核基因调节蛋白）的靶标搜索提供重要且普遍的见解。该项目中获得的实验数据将立即与 Yaakov Levy 博士的小组（以色列魏茨曼科学研究所）共享和讨论，该小组一直在研究 DNA 结合蛋白促进目标定位的理论和计算方面。这项国际合作将整合实验和理论方法，以对目标搜索过程产生更现实的看法。 更广泛的影响：通过整合研究和教育，该项目旨在建立一个制度环境，使学生和研究人员可以在其研究中有效利用核磁共振波谱。教育部分为德克萨斯大学医学分部 (UTMB) 的研究生和研究人员提供了学习 NMR 作为研究工具的机会。该项目的首席研究员 (PI) 将设计基于 Mathematica 的 NMR 波谱学教学大纲，并启动一门选修课程来教授 NMR 波谱学的实际方面，展示实际的数据收集和分析。此外，PI 将继续组织 NMR 研讨会，作为分享知识和工具的有效手段。该项目的教育部分包括高中生的科学教育以及国际学生与合作者实验室的交流。通过 UTMB 的 HSSRP 项目，高中生参与了该项目的研究。这为高中生提供了一个引人入胜的项目，他们结合各自在生物学、化学、物理和数学方面的知识，在专业环境中进行科学研究。高中生将主要由参与该项目的研究生监督，这将为 UTMB 学生提供在没有常规本科课程的机构任教的绝佳机会。国际学生交流将为不同文化背景的研究生提供机会，让他们了解更广阔的科学视野的重要性，因为两个群体都在通过完全不同且互补的方法研究同一课题。