STRUCTURE/FUNCTION OF NUCLEIC ACIDS

描述：通过两个磷酸二酯从前mRNAs中去除内含子 剪接体催化的切割反应。与大多数酶不同 复合体，剪接体由100多个组分组成 在每个衬底周围从头组装。基材种类繁多， 大小和序列，包含许多潜在的剪接位点，但正确 剪接位点总是被选择的。剪接似乎是从根本上 RNA催化的，因为相同的化学反应在体外发生 在仅RNA反应中，自剪接第二组内含子。剪接体是 由五个单链RNA和一个前信使核糖核酸有序相互作用形成 底物。剪接体组装的动态过程是由一种 依赖于RNA的ATPase家族，至少部分解释了 剪接体反应中对三磷酸腺苷水解的要求。在这个范围内 框架本申请的具体目标如下：i) 利用酿酒酵母系统，寻找新的剪接体蛋白 将会被执行。这些蛋白质将通过一个大型的 小核糖核酸和剪接体的遗传筛选及特征分析 中间体，ii)至少五个依赖剪接体RNA的ATPase介导 剪接体组装的不同步骤，但在任何情况下都不是功能 对这种由ATP驱动的反应的理解。因此，计划 确定大肠杆菌中DbpA蛋白的三维结构， 这个蛋白质家族的原型，其靶标是73个核苷酸 23S rRNA片段。此外，两种依赖于RNA的ATPase的作用 来自参与预剪接体组装的酵母Prp5和Prp22， 其作用是从剪接体中释放剪接的mRNA产物 已确定，iii)关于小分子RNA在催化中的作用， 需要回答的主要问题是交互在哪些方面有效 第一次反应的时间，以及发生了什么结构性变化 允许第二种反应。在剪接体组装中执行这些操作 互动发生了吗？什么是RNA-蛋白质相互作用调节的形成 和活动地点的稳定吗？使用体外重组技术 系统Abelson博士将探索化学和结构要求 通过筛选特定碱基的改变和缺失来实现功能的小分子RNA 类比和主干扰动。通过照片交联法， 剪接体中的RNA-RNA和RNA-蛋白质的相互作用将被识别。 他将确定是否在以下连续步骤中形成交叉链接 通过使用阻断a的突变体提取物进行剪接体组装 特定的步骤。

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.