STRUCTURE AND FUNCTION OF THE GROUP II INTRON RIBOZYME

本项目将阐明第二组的结构和机制 内含子，一种催化的RNA分子(或核酶)， 在酶学和结构上都没有特征。第二类内含子自身 剪接是高等植物新陈代谢的中心，它分享 真核细胞前信使核糖核酸剪接装置的机制特征。 出于这个原因，它可以构成化学物质和 参与所有真核RNA剪接的结构元件。因为. 它与我们自己的RNA加工形式相似，即第二组内含子 可能提供了对残留的RNA催化的洞察 在高等生物体中很重要。活性部位和底物专一性 第二组内含子的那些看起来不同于其他核酶， 因此，它的特性将推动已知的RNA反应性的极限 并增加应用核酶所需的知识基础 在基因治疗方面。这项研究的第一个目标是比较 已知的第二组自剪接内含子的反应性，以了解哪一个是 最有动力的效率，并了解第二组的差异 形态会影响它们的反应活性。两个内含子的效率- 分步反应和单步反应将通过监测 32P标记的RNA转录本的外显子连接和切割。基于 本研究中，一个特定的第II类内含子将成为进一步研究的重点 调查。第二个目标是确定 的催化必需结构域1和5之间的三级相互作用 第二组内含子。这些相互作用是RNA-RNA的不寻常形式 接触可能类似于核糖2；-OH-碱接触 在四膜虫核酶中鉴定。试管内选择技术 并且RNA足迹将有助于鉴定关键的2‘-OH， 适当的叔基所需的磷酸盐和碱性官能团 结构形成。第三个目标是将第二组 从单分子物种到多个翻转的自剪接RNA 适用于第一步详细酶学分析的核酶 拼接。这是第二组剪接的第一步，即 可能是最不寻常的。多翻转核酶将是 通过将内含子片段分成催化必需的结构域而产生 1和5，通过转录结构域5-结构域6亲核辅助因子 合成类似5‘外显子-内含子的核酶“底物” 边界序列。多组分核酶将在 产生单个速率和结合常数的条件 活性中心和催化机理的描述。动能 然后将使用框架来测试模型的第二组内含子反应 通过对个体进行诱变和官能团取代 组件。该框架还将促进第四个目标： 建立第二组内含子亚域和小内含子之间的类比 真核剪接器的核RNA(SnRNAs) 剪接体)。将插入U和U2单链RNA以取代结构域5和 结构域6的一部分，作为多周转结构的反应。 催化功能的交换将为RNA提供确凿的证据 核糖核蛋白剪接体内的催化作用。

This project will elucidate the structure and mechanism of the Group II intron, a catalytic RNA molecule (or ribozyme) which remains enzymologically and structurally uncharacterized. Group II intron self- splicing is central to the metabolism of higher plants and it shares mechanistic features with the eukaryotic pre-mRNA splicing apparatus. For this reason, it may constitute a model for the chemical and structural elements involved in all eukaryotic RNA splicing. Because of its similarities to our own forms of RNA processing, the Group II intron may provide insight into the remnants of RNA catalysis which remains important in higher organisms. The active site and substrate specificity of the Group II intron appear different from those of other ribozymes, so its characterization will push back the limits of known RNA reactivity and increase the base of knowledge required for application of ribozymes in gene therapy. The first objective of this study is to compare the reactivity of known self-splicing Group II introns to learn which one is most kinetically efficient and to understand how differences in Group II morphology affect their reactivity. Efficiency of the introns in two- step and single-step reactions will be compared by monitoring rates of exon ligation and cleavage from 32p-labeled RNA transcripts. Based on this study, a particular Group II intron will become the focus of further investigation. The second objective is to determine the location of tertiary interactions between catalytically essential Domains 1 and 5 of the Group II intron. These interactions are unusual forms of RNA-RNA contact potentially analogous to the ribose 2;-OH--base contacts I identified in the Tetrahymena ribozyme. Techniques of in-vitro selection and RNA footprinting will aid in the identification of critical 2'- OH, phosphate and base functionalities which are required for proper tertiary structure formation . The third objective is to convert the Group II self-splicing RNA from a unimolecular species to a multiple-turnover ribozyme suitable for detailed enzymological analysis of the first step of splicing. It is the first step of Group II splicing which is potentially the most unusual. The multiple-turnover ribozyme will be created by fragmenting the intron into catalytically essential Domains 1 and 5, by transcription of a Domain 5-Domain 6 nucleophilic cofactor and synthesis of a ribozyme "substrate" analogous to 5'exon-intron boundary sequences. The multicomponent ribozyme will be analyzed under conditions which will yield the individual rate and binding constants descriptive of the active site and catalytic mechanism. The kinetic framework will then be used to test models for Group II intron reactivity through mutagenesis and functional group substitution on the individual components. The framework will also facilitate a fourth objective: to establish analogies between subdomains of the Group II intron and small nuclear RNAs (snRNAs) of the eukaryotic splicing apparatus (the spliceosome). U and U2 snRNAs will be inserted in place of Domain 5 and a portion of Domain 6 in reaction of the multiple turnover construct. Exchange in catalytic function would provide firm evidence for RNA catalysis within the ribonucleoprotein spliceosome.