STRUCTURE AND FUNCTION OF THE GROUP II INTRON RIBOZYME

II组内含子核酶的结构和功能

• 批准号: 2022797
• 负责人: Anna Marie Pyle
• 金额: $ 27.08万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022797
• 关键词: DNA footprinting; RNA splicing; active sites; catalyst; chemical structure function; enzyme substrate analog; gel mobility shift assay; genetic transcription; introns; nucleic acid sequence; protein folding; radiotracer; ribozymes; small nuclear RNA; stereochemistry

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.

该项目将阐明II组的结构和机制 内含子，催化RNA分子（或核酶），保留 酶学和结构上没有特征。 II组内含子自我 剪接对于高等植物的代谢至关重要，IT共享 真核前mRNA剪接设备的机械特征。 因此，它可能构成化学物质的模型和 所有真核RNA剪接涉及的结构元素。 由于 它与我们自己的RNA处理形式的相似之处，II组内含子 可能会洞悉RNA催化的残留物 在较高的生物体中很重要。 活性位点和底物特异性 II组内含子的外观与其他核酶的内含子不同， 因此其表征将推迟已知RNA反应性的限制 并增加应用核酶所需的知识基础 在基因疗法中。 这项研究的第一个目的是比较 已知的自剪拼式组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II组的差异如何 形态会影响其反应性。 内含子在两种的效率 通过监视速率，将比较步骤和单步反应 从32p标记的RNA转录物中的外显子连接和裂解。 基于 这项研究，特定的II组内含子将成为进一步的重点 调查。 第二个目标是确定 催化性的必需域1和5之间的三级相互作用 II组内含子。 这些相互作用是RNA-RNA的异常形式 接触可能类似于核糖2; -oH-基键触点i 在四膜膜核酶中鉴定。 体外选择的技术 RNA足迹将有助于识别关键2'-哦， 适当三级所需的磷酸盐和基础功能 结构形成。 第三个目标是转换II组 从单分子物种到多弯曲的自剪接RNA 核酶适用于第一步的详细酶学分析 剪接。 这是II组拼接的第一步，是 可能是最不寻常的。 多重核酶将 通过将内含子碎裂成催化的必需域而创建 1和5，通过域5域6亲核辅因子的转录 核酶“底物”的合成类似于5'EXON-INTRON 边界序列。 多组分核酶将在 将产生个体速率和结合常数的条件 活性位点和催化机制的描述。 动力学 然后，框架将用于测试II组内含子反应性的模型 通过对个体的诱变和功能群体的替代 成分。 该框架还将促进第四个目标： 在II组内含子和小的子域之间建立类比 真核剪接设备的核RNA（SNRNA）（ 剪接体）。 U和U2 snrnas将代替域5插入 域6的一部分在多个周转构建体的反应中。 催化功能的交换将为RNA提供牢固的证据 核糖核蛋白剪接体内的催化。