NUCLEIC ACID STRUCTURE AND REACTIVITY

该项目的主要目标是了解特定的RNA 结构有助于RNA催化。 主要实验 系统将继续是自我拼接IVS（干预 四膜虫的核糖核酸酶（RNA酶） IVS 其他目标是制定一套序列- 可用作RNA切割工具的特异性RNA切割酶 分子生物学和探索RNA催化的一般性 新方向 具体目标如下：（1）获得三维视图 四膜虫IVS RNA的活性位点的变化， 底物结合。 方法将包括化学改性， 通过活性位点定向抑制剂切割RNA，和UV 交联 (2)识别参与底物的核苷酸- 使用位点特异性和随机诱变的结合和催化。 催化剂的三维结构的特定模型 核心将被测试。 (3)进一步探讨了 核酶作为序列特异性核糖核酸内切酶。 20个活跃的- 具有改变的底物特异性的位点变体将被 表征了 (4)验证核mRNA剪接是在 至少部分由小核RNA催化， 酿酒酵母系统。 (5)验证mRNA 在某些情况下，稳定性可能取决于 自切割位点。 四膜虫IVS RNA提供了一个异常适合的系统， 了解RNA中的结构-功能关系， 一般的生物催化。 很可能，许多他 研究结果将适用于其他系统，其中RNA是在 催化，包括其他RNA加工反应和蛋白质 合成.

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.