PROBING RNA METAL BINDING WITH SPECIFICALLY LABELED RNA

我们将使用15N/13C特异性标记的RNA片段的15N NMR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，为此我们将准备专门标记的片段来研究各种金属阳离子的结合。1. 一些小基序含有不同的GU和GA对组合，其中一些含有其他非沃森-克里克对和/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不同金属离子的多重实验。2. 锤头酶和发夹酶是相对较小的核酶（40 - 50nt），可通过化学合成获得。它们需要金属阳离子才能发挥作用，并且在治疗和诊断应用方面显示出很大的希望。3. I族和II族内含子要大得多（400 - 600nt），需要金属阳离子才能形成活性三级结构并进行催化。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，并且需要金属阳离子来完成。一个特别的焦点将是三螺旋连接，它通常在形成必要的核糖体结构和结合金属和蛋白质方面发挥重要作用。

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.