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

该奖项是根据2009年美国复苏和再投资法案(公法111-5)资助的。DNA结合蛋白的基因调控对生命系统至关重要。这种蛋白质通过与特定的DNA序列结合来控制基因的表达，这种联系必须是快速的，才能对细胞信号做出快速反应。该项目的总体目标是在大量非特异性DNA存在的情况下，对目标搜索过程有一个物理上的了解，从而使DNA结合蛋白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侧重于通过具有多个锌指DNA结合结构域(DBD)的基因调控蛋白进行靶标搜索的结构、动态和动力学方面。利用核磁共振波谱，本项目将以含有三个锌指DBD的Zif268蛋白为模型系统来分析目标搜索。这种蛋白是一种可诱导的基因调控蛋白，参与神经元的可塑性和记忆形成，必须有效地找到其靶DNA序列，以介导细胞对神经元信号的快速反应。Zif268蛋白是研究目标搜索过程的理想模型，因为它已经在生化、结构和功能方面得到了广泛的表征，并且对特定和非特定的DNA复合体都表现出了良好的核磁共振谱。本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)所谓的Zif268蛋白的直接转移机制有多有效3)在高DNA浓度下滑动机制对目标搜索的相对贡献为了回答这些问题，将使用他们先前研究中开发的各种核磁共振方法。这个项目将为锌指蛋白的靶标搜索提供重要和一般性的见解，锌指蛋白是真核基因调控蛋白中含量最丰富的一类。在该项目中获得的实验数据将立即与Yaakov Levy博士的小组(以色列魏兹曼科学研究所)分享和讨论，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的问题。这一国际合作将把实验和理论方法结合起来，以产生对目标搜索过程更现实的看法。更广泛的影响：通过整合研究和教育，该项目旨在建立一个机构环境，使学生和研究人员能够在他们的研究中有效地利用核磁共振波谱。教育部分提供了机会，让德克萨斯大学医学院(UTMB)的研究生和研究人员可以将核磁共振作为一种研究工具来学习。该项目的首席研究员(PI)将设计基于数学的核磁共振光谱学教学大纲，并开设一门选修课，教授核磁共振光谱学的实际方面，演示实际的数据收集和分析。此外，国际和平研究所将继续组织核磁共振研讨会，作为交流知识和工具的有效手段。该项目的教育部分包括对高中生的科学教育和与合作者实验室的国际学生交流。通过UTMB的HSSRP计划，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，在这个项目中，他们将自己在生物、化学、物理和数学方面的独立知识结合起来，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指导，这将给UTMB的学生提供一个在没有正规本科课程的机构任教的绝佳机会。国际学生交流将为具有不同文化背景的研究生提供机会，让他们学习更广泛的科学视野的重要性，因为两个群体中的每个人都在以完全不同和互补的方法研究同一主题。

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.