MECHANISM AND STRUCTURE OF A NOVEL CATALYTIC RNA

虽然最著名的是自剪接能力，但I组内含子也可以 催化其他反应，如连接寡核苷酸， 切割内含子RNA的环化和3‘端剪接的水解性 地点。Didymium rDNA内含子DiSSU1含有两个不同的核酶， GIR1和GIR2。GIR1是一个新的I族内含子，因为它是第一个 核酶，其假定的催化功能是水解，是 迄今确定的最小(约150新台币)。三个不同的但 补充方法、动力学、体外选择和x射线 结晶学，将用于表征GIR1。最优的条件 催化作用和水解动力学将被确定。离体 将使用选择方法来选择GIR1样核酶 提高了催化效率。此外，为了进一步 了解自拼接所需的最小结构特征， GIR1将被转化为自剪接核酶，并在 体外选择。这些方法将为比较提供序列数据 序列分析和建模，提供了一种更好的获得 用于结晶试验的同质核糖核糖核酸池。最后， Didymium GIR1、体外筛选的GIR1核酶、AS 以及Naegleria GIR1将启动。加深了对 GIR1的催化机理和模具结构要求 将促进改变或改进的I组核酶的设计 用于治疗的活动。

Although best known for self-splicing ability, groups I introns can also catalyze other reactions such as ligation of oligonucleotides, circularization of excised intron RNA, and hydrolysis at the 3' splice site. The Didymium rDNA intron DiSSU1 contains two distinct ribozymes, GIR1 and GIR2. GIR1 is a novel group I intron in that it is the first ribozyme whose putative catalytic function is hydrolysis, and is the smallest (approximately 150 nt) identified to date. Three different but complementary methods, kinetics, in vitro selection, and x-ray crystallography, will be used to characterize GIR1. Conditions for optimal catalysis and the kinetics of hydrolysis will be determined. In vitro selection methods will be used to select for GIR1-like ribozymes with increased catalytic efficiency. In addition, in order to further understand the minimal structural features required for self-splicing, GIR1 will be converted into a self-splicing ribozyme, and subjected to in vitro selection. These methods will provide sequence data for comparative sequence analysis and modeling, and provide a better method for obtaining homogeneous pools of RNA for use in crystallization trials. Lastly, crystallization of Didymium GIR1, in vitro selected GIR1 ribozymes, as well as Naegleria GIR1 will be initiated. Increased understanding of the catalytic mechanism and die structural requirements for hydrolysis by GIR1 will facilitate the design of group I ribozymes with altered or improved activity for therapeutic use.