STRUCTURE/FUNCTION OF NUCLEIC ACIDS

核酸的结构/功能

• 批准号: 2021972
• 负责人: JOHN N ABELSON
• 金额: $ 67.38万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2021972
• 关键词: Escherichia coli; RNA splicing; Saccharomyces cerevisiae; active sites; adenosinetriphosphatase; catalyst; enzyme mechanism; enzyme substrate complex; high performance liquid chromatography; introns; laboratory mouse; laboratory rabbit; mass spectrometry; messenger RNA; microorganism genetics; nucleic acid structure; protein biosynthesis; protein purification; protein structure function; small nuclear RNA; spliceosomes; temperature sensitive mutant

DESCRIPTION: Introns are removed from pre-mRNAs via two phosphodiester cleavage reactions catalyzed by spliceosomes. In contrast to most enzyme complexes, the spliceosome consists of more than one hundred components and is assembled de novo around each substrate. The substrates are diverse in size and sequence, containing many potential splice sites, yet the correct splice sites are invariably chosen. Splicing appears to be fundamentally RNA-catalyzed because the same chemical reactions occur in vitro for self-splicing Group II introns, in an RNA-only reaction. Spliceosome is formed through an ordered interaction of five snRNAs and a pre-mRNA substrate. The dynamic process of spliceosome assembly is catalyzed by a family of RNA -dependent ATPases explaining, at least in part the requirement for ATP hydrolysis in the spliceosomal reaction. Within this framework the specific aims of this application are as follows: i) employing the S. Cerevisiae system, a search for new spliceosomal proteins will be carried out. The proteins will be identified through a large genetic screen and by characterization of snRNAs and spliceosomal intermediates, ii) at least five spliceosomal RNA-dependent ATPases mediate different steps in spliceosome assembly, and yet in no case is the function of the ATP-driven reaction understood. Therefore, it is planned to determine the three dimensional structure of the DbpA protein from E. coli, a prototype of this family of proteins, whose target is a 73-nucleotide fragment of 23S rRNA. In addition, the roles of two RNA-dependent ATPases from yeast Prp5, which participates in pre-spliceosome assembly, and Prp22, which acts to release spliced mRNA product from the spliceosome will be identified, iii) concerning the roles of the snRNAs in catalysis, the principle questions to be answered are what interactions are in effect at the time of the first reaction, and what structural changes take place to allow the second reaction. When in spliceosome assembly do these interactions occur? What RNA-protein interactions mediate the formation of and stabilization of the active site? Using an in vitro reconstitution system Dr. Abelson will explore the chemical and structural requirements of functional snRNAs by screening specific base changes and deletions, base analogs, and backbone perturbations. By photo cross-linking methods, RNA-RNA and RNA-protein interactions in the spliceosome will be identified. He will determine whether cross-links are formed at successive steps of spliceosome assembly through the use of mutant extracts that block a particular step.

描述：内含子通过两个磷酸二酯从前体mRNA中去除 剪接体催化的裂解反应。 与大多数酶相比， 复合物中，剪接体由一百多个组分组成， 在每个基底周围重新组装。 基质是多种多样的， 大小和序列，包含许多潜在的剪接位点，但正确的 总是选择剪接位点。 剪接似乎从根本上 RNA催化，因为相同的化学反应发生在体外， 在仅RNA反应中自我剪接II组内含子。 剪接体是 通过五个snRNA和一个前mRNA的有序相互作用形成 衬底 剪接体组装的动态过程是由一个 依赖RNA的ATP酶家族至少部分解释了 剪接体反应中ATP水解的需要。 在这 本申请的具体目的如下：i） 采用S. Cerevisiae系统，寻找新的剪接体蛋白 将被执行。 这些蛋白质将通过一个大的 通过snRNA和剪接体的表征进行遗传筛选 ii）至少五种剪接体RNA依赖性ATP酶介导 在剪接体组装中的不同步骤，但在任何情况下， ATP驱动的反应。 因此计划 确定了E.大肠杆菌， 这个蛋白质家族的原型，其目标是一个73个核苷酸的 23S rRNA的片段。 此外，两种RNA依赖性ATP酶的作用 来自酵母Prp5，其参与前剪接体组装，和Prp22， 其作用是从剪接体释放剪接的mRNA产物， iii）关于snRNA在催化中的作用， 需要回答的主要问题是， 第一次反应的时间，以及发生了什么结构变化， 允许第二个反应。 在拼接组装时， 互动发生？ 什么RNA-蛋白质相互作用介导了 和活性部位的稳定性 使用体外重建 Abelson博士将探讨系统的化学和结构要求， 通过筛选特异性碱基改变和缺失， 类似物和主链扰动。 通过光交联方法， 将鉴定剪接体中的RNA-RNA和RNA-蛋白质相互作用。 他将确定交联是否在以下连续步骤中形成： 通过使用突变体提取物进行剪接体组装， 特别的一步。