Monovalent metal ion binding sites in nucleic acids

描述（由申请人提供）：本提案的总体目标是 为了鉴定和表征其中的特定单价金属离子结合位点， RNA的三级结构金属离子在折叠过程中起着重要的辅助因子的作用 的RNA。这些金属离子结合位点的鉴定几乎集中在 仅限于二价离子，如Mg 2+，它可以发挥结构上的作用， 和RNA中的催化作用。虽然先前的研究强调， 二价离子，许多具有基本生物学功能的RNA明确需要 钾或钠阳离子折叠和/或促进体外催化。的 核糖体（蛋白质合成）、II组内含子（内含子剪接）和RNase P（tRNA成熟）都需要0.2 - 2 M的单价离子来催化 他们各自的反应，这一要求不能被克服高 二价离子的浓度。具体和功能上的可能性 RNA内的相关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，它们尚未被探索。 该提案概述了一系列新的生物化学和生物物理方法 来解决这个问题。这些方法利用一价铊（Tl+）， 重金属阳离子，化学性质与生理碱相似 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，但它有属性，将是有价值的 用于分析与RNA结合的单价金属离子，包括优异的 NMR感受性和增强的配位硫的能力。着力 针对四个研究目标，利用化学性质， 用于RNA中K+或Na+结合位点的研究。这些目标是： (i)在所有三种主要类型的金属离子中鉴定K+金属离子位点。 大的催化RNA;（ii）确定结构和生物活性 核糖体移码中单价离子结合位点的相关性 假结;（iii）测定两个单价金属离子的结合作用 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;（iv） ~（205）TlNMR直接检测一价~（205）Tl-NMR方法的研究进展 使用DNA G-四重体作为模型系统与核酸结合的金属离子; (iv)~（205）Tl NMR研究金属离子与RNA的结合 包括催化RNA、假结和信号识别的系统 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.