DEFINITION OF THE CATALYTIC SITE OF THE GROUP II INTRON

II 组内含子催化位点的定义

• 批准号: 2332002
• 负责人: KEVIN A JARRELL
• 金额: $ 20.4万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2332002
• 关键词: RNA splicing; active sites; chemical cleavage; chemical kinetics; crosslink; endonuclease; enzyme mechanism; enzyme substrate; esterification; introns; nucleic acid sequence; precursor mRNA; ribozymes; site directed mutagenesis

DESCRIPTION: Human diseases that result from defects in intron splicing could be treated with therapeutics that control specific intron splicing reactions. This is not currently possible because we lack a sufficiently detailed understanding of the mechanism of human pre-mRNA intron splicing. Pre-mRNA splicing is catalyzed by the spliceosome, which consists of 60 different proteins and five RNAs. The physical complexity of the spliceosome has hampered studies of splicing. The spliceosome is not required for the splicing of certain fungal introns that are related to human pre-mRNA introns, the self-splicing group II introns. Studies of group II intron splicing are relevant to studies of pre-mRNA splicing because group II intron splicing occurs by the same mechanism, involving two sequential transesterification reactions, used by the spliceosome. In summary, the self-splicing group II intron is a simple model system for studies of human pre-mRNA splicing. Definition of the catalytic core of the group II intron will help define the catalytic core of the spliceosome. These studies will provide basic information relevant to applied research, aimed at the discovery of novel therapeutics. A fundamental question is whether group II introns, and the spliceosome, use a single active site or two active sites to catalyze the two reactions of splicing. The investigator has developed a system that will allow him to determine the number of group II intron active sites. These studies will focus on two endonucleolytic cleavage reactions. One cleavage mimics the first reaction of splicing, the other mimics the second reaction. If the ribozyme has one active site, the same sequences should be required for both cleavage reactions. A particular intron sequence, domain 5, is required for the first reaction of splicing. Whether domain 5 is required for the second reaction is unknown. One reason for this lack of information is that mutations that block the first reaction also block the second, by default, since splicing occurs in two sequential reaction steps. To circumvent this problem the investigator will produce the two RNAs that would result if introns that lack domain 5 could complete the first step of splicing. The ability of these RNAs to complete the second reaction will be tested. These studies will determine whether domain 5 is required for the second reaction. Domain 5 binds tightly to a site located in another region of the intron. This interaction probably forms a key component of the active site that catalyzes the first reaction. Binding occurs by a novel tertiary interaction. The binding partner of domain 5 has not been located. The 3-dimensional structure of this interaction may be modeled using cross- linking, iron-EDTA and mutagenesis. These studies will characterize a novel RNA-RNA interaction that is likely to be essential for human pre- mRNA splicing. By defining the key ribozyme sequences that catalyze the two steps of group II splicing these studies will provide significant new information relevant to the design of therapeutic agents that control particular intron splicing reactions.

描述：由内含子剪接缺陷引起的人类疾病 可以用控制特定内含子剪接的疗法进行治疗 反应。 目前这是不可能的，因为我们缺乏足够的 详细了解人类mRNA前体内含子机制 拼接。 前 mRNA 剪接由剪接体催化，剪接体 由 60 种不同的蛋白质和 5 种 RNA 组成。 物理方面 剪接体的复杂性阻碍了剪接的研究。 这 某些真菌内含子的剪接不需要剪接体 与人类前 mRNA 内含子相关的自剪接组 II 内含子。 II组内含子剪接的研究与研究相关 前 mRNA 剪接的影响，因为 II 组内含子剪接是通过相同的方式发生的 机制，涉及两个连续的酯交换反应，使用 通过剪接体。 综上所述，自剪接 II 族内含子是 用于研究人类前体 mRNA 剪接的简单模型系统。 定义 II组内含子催化核心的研究将有助于定义 剪接体的催化核心。 这些研究将提供基础 与应用研究相关的信息，旨在发现新颖的 疗法。 一个基本问题是第二组内含子和剪接体是否 使用单个活性位点或两个活性位点来催化两种 剪接反应。 研究人员开发了一个系统 将使他能够确定 II 组内含子活性位点的数量。 这些研究将集中于两种核酸内切裂解反应。 一 切割模仿剪接的第一个反应，另一个模仿剪接 第二个反应。 如果核酶有一个活性位点，则同样 两个裂解反应都需要序列。 第一个需要特定的内含子序列（结构域 5） 拼接反应。 第二个是否需要域5 反应未知。 缺乏信息的原因之一是 阻止第一个反应的突变也会阻止第二个反应，通过 默认，因为剪接发生在两个连续的反应步骤中。 到 为了规避这个问题，研究人员将产生两种 RNA 如果缺少结构域 5 的内含子能够完成第一步，则会产生结果 的拼接。 这些RNA完成第二个反应的能力 将受到测试。 这些研究将确定域 5 是否是 第二次反应所需。 结构域 5 与位于内含子另一个区域的位点紧密结合。 这种相互作用可能构成活性位点的关键组成部分 催化第一反应。 结合通过新颖的三级发生 相互作用。 结构域 5 的结合配偶体尚未定位。 这 这种相互作用的 3 维结构可以使用交叉模型来建模 连接、铁-EDTA 和诱变。 这些研究将描述一个 新型RNA-RNA相互作用可能对于人类预防至关重要 mRNA 剪接。 通过定义催化以下两个步骤的关键核酶序列 第二组拼接这些研究将提供重要的新信息 与控制特定的治疗剂的设计相关 内含子剪接反应。