Monovalent metal ion binding sites in nucleic acids

描述(申请人提供)：本建议书的总体目标是 鉴定和表征特定的单价金属离子结合部位 RNA三级结构。金属离子在折叠过程中起着重要的辅助作用 RNAs。这些金属离子结合部位的鉴定几乎都是关注焦点。 仅限于二价离子，如镁离子，它既可以起到结构上的作用 以及在RNA中的催化作用。虽然之前的研究强调 二价离子，许多具有基本生物功能的RNA明确要求 钾或钠离子在体外折叠和/或促进催化作用。这个 核糖体(蛋白质合成)、第二组内含子(内含子剪接)和核糖核酸酶 P(tRNA成熟)都需要0.2到2M的单价离子来催化 他们各自的反应，而这一要求是不能通过高 二价离子浓度。特定的和功能上的可能性 RNA中相关的单价金属离子结合位点在很大程度上 由于缺乏识别它们的方法，未被探索。 这份提案概述了一系列新的生化和生物物理方法。 来解决这个问题。这些方法利用一价铊(Tl+)，a 化学性质与生理碱相似的重金属阳离子 金属钾(K+)和钠(Na+)。Tl+可以有效地模拟 这些阳离子的生物作用，但它的性质将是有价值的 用于分析单价金属离子与RNA的结合，包括优秀的 核磁共振感受性和增强的硫配位能力。我们将努力 针对四个利用化学特性的研究目标 用于研究RNA中的K+或Na+结合部位。这些目标是： (I)确定所有三大类金属离子中的K+金属离子位置 大分子催化RNA；(Ii)结构和生物学性质的测定 核糖体移框中单价离子结合部位的相关性 假结；(Iii)两种单价金属离子结合作用的测定 位于信号识别颗粒的蛋白质-RNA核心内的位点， 生物学中最保守的核糖核蛋白相互作用之一； 直接检测单价化合物的~(205)Tl核磁共振研究进展 以DNA G-四元组为模型体系研究金属离子与核酸的结合； (四)利用~(205)Tl核磁共振研究复杂RNA中单价金属离子的结合 包括催化RNA、伪结和信号识别在内的系统 粒子。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to identify and characterize specific monovalent metal ion binding sites within RNA tertiary structures. Metal ions act as essential cofactors in the folding of RNAs. The identification of these metal ion binding sites has focused almost exclusively on divalent ions, such as Mg2+, which can play both a structural and a catalytic role within RNAs. While previous research has emphasized divalent ions, many RNAs with essential biological functions explicitly require potassium or sodium cations to fold and/or to promote catalysis in vitro. The ribosome (protein synthesis), the group II intron (intron splicing), and RNase P (tRNA maturation) all require between 0.2 and 2 M monovalent ion to catalyze their respective reactions, and this requirement cannot be overcome by high concentrations of divalent ion. The possibility of specific and functionally relevant monovalent metal ion binding sites within RNA has been largely unexplored, due to a lack of methods with which to identify them. This proposal outlines a new series of biochemical and biophysical approaches to address this problem. These approaches utilize monovalent thallium (Tl+), a heavy metal cation with chemical properties similar to the physiological alkali metals potassium (K+) and sodium (Na+). Tl+ can effectively mimic the biological action of these cations, yet it has properties that will be valuable for the analysis of monovalent metal ion binding to RNA, including excellent NMR receptivity and an enhanced ability to coordinate sulfur. Efforts will be directed toward four research objectives that utilize the chemical properties of TI+ for the study of K+ or Na+ binding sites in RNA. These objectives are: (i) Identification of a K+ metal ion site within all three major classes of large catalytic RNA; (ii) Determination of the structural and biological relevance of a monovalent ion binding site within the ribosomal frameshifting pseudoknot; (iii) Determination the role of two monovalent metal ion binding sites located within the protein-RNA core of the signal recognition particle, one of the most conserved ribonucleoprotein interactions in biology; (iv) Development of 205Tl NMR as a method for the direct detection of monovalent metal ion binding to nucleic acids using the DNA G-quartet as a model system; (iv) Utilization 205Tl NMR to study monovalent metal ion binding in complex RNA systems including catalytic RNAs, pseudoknots, and the signal recognition particle.