MECHANISM AND STRUCTURE OF A NOVEL CATALYTIC RNA

新型催化RNA的机制和结构

• 批准号: 2415074
• 负责人: EVELYN JABRI
• 金额: $ 2.44万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2415074
• 关键词: RNA splicing; X ray crystallography; chemical kinetics; crystallization; enzyme mechanism; enzyme structure; hydrolysis; introns; nucleic acid sequence; nucleic acid structure; ribozymes

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.

虽然最出名的自我剪接能力，I组内含子也可以 催化其它反应如寡核苷酸的连接， 切下的内含子RNA的环化和3'剪接处的水解 绝佳的价钱Didymium rDNA内含子DiSSU1含有两种不同的核酶， 女孩1和女孩2。GIR1是一个新的I组内含子，因为它是第一个 核酶，其假定的催化功能是水解，并且是 最小的（约150 nt）确定的日期。三个不同的，但 互补方法、动力学、体外选择和X射线 晶体学，将用于表征GIR1。最优条件 将测定催化和水解动力学。体外 选择方法将用于选择GIR1样核酶， 提高催化效率。此外，为了进一步 了解自我拼接所需的最小结构特征， GIR1将被转化为一种自我剪接的核酶，并在 离体选择这些方法将提供序列数据用于比较 序列分析和建模，并提供了一个更好的方法，获得 用于结晶试验的RNA的均质池。最后， Didymium GIR 1的结晶，体外选择的GIR 1核酶， 以及耐格里属GIR1将被启动。进一步了解如何 GIR 1水解的催化机理和模具结构要求 将有助于设计具有改变或改善的 活性用于治疗用途。