NUCLEIC ACID STRUCTURE AND REACTIVITY

核酸结构和反应性

• 批准号: 3484628
• 负责人: THOMAS ROBERT CECH
• 金额: $ 14.66万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-08-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3484628
• 关键词: Escherichia coli; RNA biosynthesis; RNA splicing; Saccharomyces; Tetrahymena; affinity labeling; autoradiography; chemical fingerprinting; chemical group; cofactor; enzyme mechanism; enzyme structure; evolution; gel electrophoresis; gene expression; genetic manipulation; genetic recombination; genetic strain; guanosine diphosphate; microorganism genetics; molecular cloning; nucleic acid hybridization; nucleic acid sequence; nucleoside analog; radiotracer; ribosomal RNA; ribozymes; temperature sensitive mutant; tissue /cell culture

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.

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。 主要实验 系统将继续是自拼接IVS（介入 四膜虫的序列）和核酶（RNA 酶）源自 IVS。 额外的目标是开发一套序列- 可用作 RNA 工具的特定 RNA 切割酶 分子生物学并探索RNA催化的普遍性 朝着新的方向。 具体目标如下： (1) 获得 3 维视图 四膜虫 IVS RNA 的活性位点，有或没有 底物结合。 方法将包括化学修饰、 通过活性位点定向抑制剂和 UV 来切割 RNA 交联。 (2) 鉴定参与底物的核苷酸- 使用位点特异性和随机诱变进行结合和催化。 催化三维结构的特殊模型 核心将被测试。 （三）进一步探索活动 核酶作为序列特异性核糖核酸内切酶。 二十个活跃的—— 具有改变的底物特异性的位点变体将是 特点。 (4) 检验核mRNA剪接的观点 至少部分由小核RNA催化 酿酒酵母系统。 (5) 检验 mRNA 的想法 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 提供了一个异常适合的系统 了解 RNA 的结构与功能关系 一般生物催化。 很可能他中的很多人 研究结果将适用于 RNA 所在的其他系统 催化，包括其他 RNA 加工反应和蛋白质 合成。