PROBING RNA METAL BINDING WITH SPECIFICALLY LABELED RNA

我们将使用15N/13C特定标记的RNA片段的15N核磁共振来识别和评估涉及碱基氮的金属结合位点。主要的焦点将是下面描述的RNA系统，我们将为其准备专门标记的片段，以研究广泛的金属阳离子的结合。1.含有GU和GA对的各种组合的几个小基序，有些与其他非Watson-Crick对和/或凸起的核苷，被认为是特定的金属结合位点。这些孤立基序的相对较小的尺寸使它们特别适合于用不同的金属离子进行多个实验。2.锤头和发夹是相对较小的核酶(40-50个核苷酸)，可通过化学合成获得。它们需要金属阳离子才能发挥作用，并在治疗和诊断应用方面显示出很大的前景。3.第一类和第二类内含子要大得多(400-600个核苷酸)，需要金属阳离子来形成活性三级结构和催化。我们将在越来越大的亚基中合成和研究这些系统，最终目标是将我们标记的片段连接到更长的RNA上。4.核糖体RNA在所有生命系统的蛋白质合成中起着核心作用，需要金属阳离子来完成这一任务。一个特别的焦点将是三螺旋连接，它通常通过形成必要的核糖体结构并与金属和蛋白质结合而发挥关键作用。

We will use 15N NMR of 15N/13C specifically labeled RNA fragments to identify and evaluate metal binding sites that involve base nitrogens. The major focus will be the RNA systems described below, for which we will prepare specifically labeled fragments to study binding of a wide range of metal cations. 1. Several small motifs containing various combinations of GU and GA pairs, some with other non-Watson-Crick pairs and/or bulged nucleosides, have been implicated as specific metal binding sites. The relatively small size of these isolated motifs makes them particularly suited to multiple experiments with different metal ions. 2. The hammerhead and hairpin are relatively small ribozymes (40 - 50 nt) that are accessible by chemical synthesis. They require metal cations to function, and show much promise for therapeutic and diagnostic applications. 3. The group I and II introns are much larger (400 - 600 nt) and require metal cations to form active tertiary structures as well as for catalysis. We will synthesize and study these systems in subunits of increasing size, with the ultimate goal of ligating our labeled fragments to much longer RNA. 4. Ribosomal RNA plays a central role in protein synthesis in all living systems, and requires metal cations to do so. A particular focus will be three-helix junctions which often play essential roles by forming the necessary ribosomal architecture and binding to metals and proteins.