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RNA分子（或核酶）， 酶学和结构上未表征。 第二组内含子自身 剪接是高等植物新陈代谢的核心， 真核前体mRNA剪接装置的机制特征。 因此，它可以构成化学品的模型， 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，第二组内含子， 可以提供对RNA催化残余的深入了解， 在高等生物中很重要。 活性位点和底物特异性 第二组内含子的结构与其他核酶不同， 所以它的特性将推回到已知的RNA反应性的极限， 并增加核酶应用所需的知识基础 基因治疗 本研究的第一个目的是比较 已知的自我剪接组II内含子的反应性，以了解哪一个是 最有效的动力学，并了解第二组中的差异 形态影响它们的反应性。 两种基因中内含子的效率- 将通过监测以下速率来比较分步和单步反应： 外显子连接和从32 p标记的RNA转录物切割。 基于 在这项研究中，一个特定的第二组内含子将成为进一步研究的重点。 调查 第二个目标是确定 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-碱基接触I 在四膜虫核酶中鉴定。 离体选择技术 而RNA足迹法将有助于鉴定关键的2 ′-OH， 磷酸盐和碱官能度，这是适当的叔 结构形成 第三个目标是将第二组 自剪接RNA从单分子种类到多翻转 核酶适用于第一步详细的酶学分析 的拼接。 这是第二组剪接的第一步， 可能是最不寻常的 多转换核酶将是 通过将内含子片段化为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亲核辅因子 和类似于5 '外显子-内含子的核酶“底物”的合成 边界层序 多组分核酶将在以下条件下进行分析： 产生单个速率和结合常数的条件 活性位点和催化机理的描述。 动力学 然后，将使用一个框架来测试II组内含子反应性的模型 通过对个体进行诱变和官能团取代， 件. 该框架还将促进第四个目标： 在第二组内含子和小内含子的亚结构域之间建立类比， 真核细胞剪接器的核RNA（snRNA） 剪接体）。 将插入U和U2 snRNA代替结构域5， 结构域6的一部分在多个翻转结构的反应中。 催化功能的交换将为RNA 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.