NUCLEIC ACID STRUCTURE AND REACTIVITY

核酸结构和反应性

• 批准号: 3484625
• 负责人: THOMAS ROBERT CECH
• 金额: $ 23.04万
• 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 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3484625
• 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; phosphorus; radionuclides; 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 结构有助于RNA催化。 主要实验 系统将继续是自动插曲的IV（干预 源自衍生自的四膜膜和核酶（RNA酶）的序列） IVS。 其他目标是开发一组序列 - 特定的RNA裂解酶，可能可作为RNA的工具有用 分子生物学并探索RNA催化的一般性 在新的方向上。 具体目的是：（1）获得3维的视图 有或没有 基板绑定。 方法将包括化学修饰， 通过活动点定向抑制剂和紫外线的裂解RNA 交联。 （2）确定涉及底物的核苷酸 - 使用位点特异性和随机诱变的结合和催化。 催化的三维结构的特定模型 核心将进行测试。 （3）进一步探索 核酶作为序列特异性内核酸酶。 二十个活动 底物特异性改变的位点变体将是 特征。 （4）测试核mRNA剪接的想法 至少部分用小核RNA催化 酿酒酵母系统。 （5）测试mRNA的想法 在某些情况下，稳定性可能取决于 自我切割站点。 四心肌IVS RNA提供了一个异常正常的系统 了解RNA中的结构功能关系 一般而言，生物催化。 他很可能 调查结果将适用于RNA中的其他系统 催化，包括其他RNA加工反应和蛋白质 合成。