Molecular Origins of Specificity in Protein-Nucleic Acid Interactions

蛋白质-核酸相互作用特异性的分子起源

• 批准号: 9514117
• 负责人: Jannette Carey
• 金额: $ 21.3万
• 依托单位: Princeton University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-15 至 1999-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9514117&HistoricalAwards=false
• 关键词: Molecular; Origins; Specificity; Protein; Nucleic

9514117 Carey The goal of this work on protein-nucleic acid interactions is to understand the molecular origins of specificity in the recognition process. How does a protein identify its target site among all available nucleic acid sites? Interest in the underlying principles of recognition motivate comparative study of different systems in order to distill out the molecular generalities that may be common among them. This work combines methods of biochemistry, biophysics, and molecular biology, with the goal of integrating structural and thermodynamic aspects of each system trying eventually to relate these aspects to the physiological role of the interaction. This proposal aims to examine molecular features responsible for specificity in two protein-nucleic acid interactions, those of bacterial arginine repressor (ArgR) and eukaryotic transcription factor IIIA (TFIIIA). These two systems share many features that recommend them as excellent examples for our studies, including the probable ability to bind both RNA and DNA and to package nucleic acids. In the case of the hexameric ArgR, important open questions concern the functional definition of the operator sequence, and the range of tolerance toward variations in both protein and DNA sequence. These issues will be approached by biochemical studies of binding to a natural operator with an extended tripartite structure, using functionally homologous but very divergent arginine repressors from E. coli and B. subtilis. Interactions of wildtype and site-directed mutant operators with each protein will be characterized by determining affinity, specificity, stoichiometry, cooperativity, and details of the DNA contacts using quantitative gel retardation and footprinting. A second aim with ArgR is to investigate a possible general regulatory role for the protein implied by some of its biochemical and physiological properties. This will be tested by using in vitro selection for ArgR-binding sequences from an E. coli genomic library. A thir d aim is to characterize a putative RNA-binding function observed in our purified samples of ArgR. To confirm that the RNA-binding moiety is ArgR, we will conduct quantitative biochemical studies of the ArgR-RNA interactions by using gel retardation and footprinting to determine affinity, specificity, stoichiometry, cooperativity, and details of the RNA contacts. In the case of TFIIIA, conflicting data in the literature indicate a need for quantitative biochemical approaches that can probe the origins of specificity in RNA binding. One hypothesis is that specificity is determined in a rapid binding equilibrium which must be studied kinetically to resolve it from subsequent particle formation. To approach this problem requires using purified recombinant components and isolated natural particles, and quantitative gel retardation and footprinting to determine the affinity, specificity, stoichiometry, cooperativity, and details of the RNA contacts in the various complexes that form during the time course of association, dissociation, competition, and exchange reaction. ***

这项关于蛋白质-核酸相互作用的工作的目标是了解识别过程中特异性的分子起源。一个蛋白质如何在所有可用的核酸位点中识别它的靶位点？对识别基本原理的兴趣激发了对不同系统的比较研究，以提炼出它们之间可能共同的分子共性。这项工作结合了生物化学、生物物理学和分子生物学的方法，目的是整合每个系统的结构和热力学方面，试图最终将这些方面与相互作用的生理作用联系起来。本研究旨在研究细菌精氨酸抑制因子（ArgR）和真核转录因子IIIA （TFIIIA）两种蛋白质与核酸相互作用的特异性分子特征。这两种系统有许多共同的特点，可以作为我们研究的优秀范例，包括可能结合RNA和DNA以及包装核酸的能力。在六聚体ArgR的情况下，重要的开放性问题涉及操作序列的功能定义，以及对蛋白质和DNA序列变化的耐受性范围。这些问题将通过与具有扩展的三方结构的天然操作符结合的生化研究来解决，使用来自大肠杆菌和枯草芽孢杆菌的功能同源但非常不同的精氨酸抑制物。野生型和定点突变操作符与每个蛋白质的相互作用将通过确定亲和性，特异性，化学计量学，协同性和DNA接触的细节来表征，使用定量凝胶延迟和足迹。ArgR的第二个目的是研究其生化和生理特性所暗示的蛋白质的可能的一般调节作用。这将通过体外选择大肠杆菌基因组文库中的argr结合序列进行测试。他们的第三个目的是表征在我们纯化的ArgR样品中观察到的假定的rna结合功能。为了确认RNA结合部分是ArgR，我们将通过凝胶阻滞和足迹法对arr -RNA相互作用进行定量生化研究，以确定RNA接触的亲和力、特异性、化学计量学、协同性和细节。在TFIIIA的情况下，文献中相互矛盾的数据表明需要定量生化方法来探测RNA结合特异性的起源。一种假设是特异性是在快速结合平衡中确定的，必须通过动力学研究来从随后的粒子形成中解决它。为了解决这个问题，需要使用纯化的重组成分和分离的天然颗粒，以及定量凝胶阻滞和足迹来确定在结合、解离、竞争和交换反应的时间过程中形成的各种复合物中的亲和性、特异性、化学计量学、协同性和RNA接触的细节。＊＊＊