STRUCTURE-FUNCTION OF RNA LOOPS AND DUPLEXES

这项工作的最终目标是了解RNA的结构和 函数关系。尽管生物学的例子表明了RNA的重要性 结构，但很少有物理研究描述这种影响 在诸如RNA：蛋白质复合体或RNA：RNA等系统上的RNA结构 互动。这里提出的工作有两个部分：1) 生物化学和生物物理参数的研究 序列特异性RNA结合蛋白--人U1a与 它的RNA底物。U1a是U1nRNP的一种蛋白，它结合 U1SnRNA茎/环II.U1a蛋白包含一个结构和 序列依赖的基序，这是许多RNA结合蛋白共同的， 所谓的“RNP保守基序”，或者，“RRM”。 了解该系统的交互作用将提供更多 信息不仅关于SnRNP的蛋白质和RNA是如何组装的， ，但也应作为此类的常规属性的模型 RNA结合蛋白。2)RNA：RNA相互作用包括碱基配对 双链和含有不成对碱基的结构基序中，三级 循环结构中的交互，以及使用 修改过的碱基。包含这些元素的RNA将在这里进行研究 为了确定它们的热力学和结构性质，再次使用 了解RNA的这些特征的目标是在 生物序列。所有的RNA都将通过核磁共振和化学和化学方法进行研究 酶探针法测定其结构，以及表征 热力学。RNA分子将通过酶法合成或 在化学上；当使用酶合成时，将有可能 掺入同位素标记的核苷酸以促进核磁共振 结构分析。

The ultimate goal of this work is to understand RNA structure and function relations. Despite biological examples of the importance of RNA structure, there are few physical studies which describe the influence of RNA structure on systems such as RNA:protein complexes or RNA:RNA interactions. The work proposed here has two components: 1) investigation of the biochemical and biophysical parameters of the association of a sequence-specific RNA binding protein, human U1A, with its RNA substrate. U1A is one of the proteins of U1 snRNP, which binds to the U1 snRNA stem/loop II. The U1A protein contains a structural and sequence-dependent motif which is common to many RNA binding proteins, the so-called "RNP conserved motif", or alternatively, "RRM". Understanding the interactions of this system will provide more information not only on how the proteins and RNA of the snRNP assemble, but should also serve as a model for the general properties of this class of RNA binding protein. 2) RNA:RNA interactions include base pairing in duplexes and in structural motifs containing unpaired bases, tertiary interactions in loop structures, and nonstandard interactions using modified bases. RNAs containing these elements will be investigated here to determine their thermodynamic and structural properties, again with the goal of understanding these features of RNA when they are found in biological sequences. All RNAs will be studied by NMR and chemical and enzymatic probing to determine their structure, as well as characterized thermodynamically. RNA molecules will be synthesized enzymatically or chemically; when enzymatic synthesis is used, it will be possible to incorporate isotopically labelled nucleotides to facilitate NMR structural analysis.