RIBOZYME-SUBSTRATE INTERACTIONS

核酶-底物相互作用

• 批准号: 2183069
• 负责人: JACK W SZOSTAK
• 金额: $ 20.87万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2183069
• 关键词: RNA; RNA splicing; SDS polyacrylamide gel electrophoresis; Tetrahymena; affinity chromatography; chemical binding; chemical kinetics; conformation; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; guanosine; hydrogen bond; mutant; nucleic acid chemical synthesis; nucleic acid sequence; nucleic acid structure; nucleoside analog; polymerase chain reaction; ribose; ribozymes; synthetic nucleotide; thiophosphate

The group I self-splicing introns, exemplified by the Tetrahymena ribosomal RNA intron, are RNA enzymes (ribozymes) that catalyze phosphodiester exchange reactions. This proposal is focused on the analysis of substrate binding by this class of RNA enzymes. Previous experiments have led to the identification of part of the guanosine binding site of this RNA enzyme. Here, methods for further defining the guanosine binding site are presented, and novel approaches to defining the RNA substrate binding site are described. These experiments use a recently described system for the in vitro genetic analysis of interactions within the ribozyme. Several enhancements of these in vitro selection and amplification methods are proposed, along with plans to use these techniques extensively in the study of ribozyme-substrate interactions. Potential enzymesubstrate interactions that are identified will be studied by the kinetic analysis of mutant enzymes in combination with chemically synthesized substrate analogue.

I组自剪接的内含子，用四膜hymena示例 核糖体RNA内含子是催化催化的RNA酶（核酶） 磷酸酯交换反应。 该提议的重点是 该类别的RNA酶对底物结合分析。 以前的 实验导致了一部分鸟嘌呤的鉴定 该RNA酶的结合位点。 在这里，用于进一步定义的方法 提出了鸟氨氨酸结合位点，并定义了新颖的方法 描述了RNA底物结合位点。 这些实验使用 最近描述了用于体外遗传分析的系统 核酶内的相互作用。 这些体外的几种增强 提出了选择和放大方法，以及使用的计划 这些技术在核酶基底的研究中广泛 互动。 鉴定出的潜在酶基底相互作用 将通过组合突变酶的动力学分析来研究 与化学合成的底物类似物。