NUCLEIC ACID STRUCTURE AND REACTIVITY

该项目的主要目标是了解特定的RNA如何 结构有助于RNA催化。主要的实验 系统将继续自拼接IVS(干预 四膜虫和核酶(RNA酶)的序列) 静脉输液系统。其他目标是开发一套序列- 特定的RNA裂解酶，可用作RNA的工具 分子生物学与探索RNA催化的共性 在新的方向上。 具体目标如下：(1)获得三维视图 对四膜虫IVS RNA活性部位的影响 底物结合。方法将包括化学修饰， 用活性位点定向的抑制物切割RNA，以及UV 交联剂。(2)确定底物中涉及的核苷酸- 使用定点和随机突变的结合和催化。 催化剂的三维结构的特殊模型 核心将接受测试。(三)进一步探索社区组织的活动方式 核酶作为一种序列特异性内切核酸酶。二十个活跃的- 具有改变底物专一性的位点变体将是 特色化的。(4)验证核转录因子剪接在 至少部分由使用小核RNA的小核RNA催化 酿酒酵母系统。(5)检验信使核糖核酸 在某些情况下，稳定性可能取决于是否存在 自裂解位点。 四膜虫IVS RNA提供了一种异常顺从的系统 学习RNA和蛋白质中的结构-功能关系 一般而言，生物催化。很可能他中的许多人 这些发现将适用于RNA所在的其他系统 催化，包括其他RNA加工反应和蛋白质 综合。

The main goal of the project is to understand how specific RNA structures contribute to RNA catalysis. The major experimental system will continue to be the self-splicing IVS (intervening sequence) of Tetrahymena and ribozymes (RNA enzymes) derived from the IVS. Additional goals are to develop a set of sequence- specific RNA cleavage enzymes that may be useful as tools for RNA molecular biology and to explore the generality of RNA catalysis in new directions. Specific aims are the following: (1) Obtain a 3-dimensional view of the active site of the Tetrahymena IVS RNA with and without the substrates bound. Approaches will include chemical modification, cleavage of the RNA by an active-site-directed inhibitor, and UV crosslinking. (2) Identify nucleotides involved in substrate- binding and catalysis using site-specific and random mutagenesis. A particular model of the 3-dimensional structure of the catalytic core will be tested. (3) Further explore the activity of the ribozyme as a sequence-specific endoribonuclease. Twenty active- site variants with altered substrate specificity will be characterized. (4) Test the idea that nuclear mRNA splicing is at least in part catalyzed by small nuclear RNAs using the Saccharomyces cerevisiae system. (5) Test the idea that mRNA stability might in some cases be determined by the presence of self-cleavage sites. The Tetrahymena IVS RNA provides an unusually amenable system for learning about structure-function relationships in RNA and biological catalysis in general. It is likely that many of he findings will be applicable to other systems where RNA is in catalysis, including other RNA processing reactions and protein synthesis.