Molecular Origins of Specificity in Protein-Nucleic Acid Interactions

9514117 Carey 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。 蛋白质如何在所有可用的核酸位点中识别其靶位点？ 对识别基本原理的兴趣激发了对不同系统的比较研究，以便提炼出它们之间可能共有的分子共性。 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，目标是整合每个系统的结构和热力学方面，最终试图将这些方面与相互作用的生理作用联系起来。 该提案旨在研究负责两种蛋白质-核酸相互作用特异性的分子特征，即细菌精氨酸阻遏物 (ArgR) 和真核转录因子 IIIA (TFIIIA)。 这两个系统具有许多共同特征，这些特征使它们成为我们研究的优秀示例，包括结合 RNA 和 DNA 以及包装核酸的可能能力。 就六聚体 ArgR 而言，重要的悬而未决的问题涉及操纵子序列的功能定义，以及对蛋白质和 DNA 序列变异的耐受范围。 这些问题将通过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阻遏物与具有扩展三方结构的天然操纵子结合的生化研究来解决。 野生型和定点突变操纵子与每种蛋白质的相互作用将通过使用定量凝胶阻滞和足迹法确定亲和力、特异性、化学计量、协同性和 DNA 接触的细节来表征。 ArgR 的第二个目标是研究该蛋白质的一些生化和生理特性所暗示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。 第三个目标是表征在我们纯化的 ArgR 样品中观察到的假定 RNA 结合功能。 为了确认 RNA 结合部分是 ArgR，我们将使用凝胶阻滞和足迹法对 ArgR-RNA 相互作用进行定量生化研究，以确定亲和力、特异性、化学计量、协同性和 RNA 接触的细节。 就 TFIIIA 而言，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，必须对其进行动力学研究，以从随后的颗粒形成中解决它。 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法，以确定在缔合、解离、竞争和交换反应过程中形成的各种复合物中RNA接触的亲和力、特异性、化学计量、协同性和细节。 ***

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. ***