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: 2872690
• 负责人: KEVIN A JARRELL
• 金额: $ 22.06万
• 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/2872690
• 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：人类疾病，导致缺陷的内含子剪接 可以用控制特定内含子剪接的疗法来治疗 反应. 这是目前不可能的，因为我们缺乏足够的 详细了解人类前mRNA内含子的作用机制 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 剪接体的复杂性阻碍了剪接的研究。 的 剪接体不是某些真菌内含子剪接所必需的 与人类前mRNA内含子相关的自我剪接组II 内含子 第二组内含子剪接的研究与 因为II组内含子剪接发生在相同的 机制，涉及两个连续的酯交换反应，使用 的《易经》。 总之，自我剪接组II内含子是一个 用于研究人前mRNA剪接的简单模型系统。 定义 II组内含子的催化核心将有助于定义 剪接体的催化核心。 这些研究将提供基础 与应用研究有关的信息，旨在发现新的 治疗学 一个基本的问题是第二组内含子和剪接体， 使用单个活性位点或两个活性位点来催化两个 剪接反应。 研究人员开发了一个系统， 将允许他确定第二组内含子活性位点的数量。 这些研究将集中在两个内切核酸裂解反应。 一 切割模仿剪接的第一反应，另一个模仿剪接的第二反应。 第二反应。 如果核酶只有一个活性位点， 两个切割反应都需要序列。 一个特殊的内含子序列，结构域5，是第一个需要的。 剪接反应。 第二个是否需要域5 反应未知。 缺乏信息的一个原因是， 阻断第一反应的突变也阻断第二反应， 默认情况下，因为剪接发生在两个连续的反应步骤。 到 为了解决这个问题，研究人员将产生两种RNA， 如果缺少结构域5的内含子可以完成第一步， 的拼接。 这些RNA完成第二次反应的能力 会得到考验 这些研究将确定域5是否是 这是第二次反应所必需的。 结构域5与位于内含子另一区域的位点紧密结合。 这种相互作用可能形成了活性位点的一个关键组成部分， 催化第一个反应。 结合发生在一个新的三级 互动 尚未找到域5的绑定伙伴。 的 3-这种相互作用的维度结构可以使用交叉- 连接、铁-EDTA和诱变。 这些研究将描述一个 新的RNA-RNA相互作用可能是人类前- mRNA剪接。 通过定义催化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 与设计控制特定疾病的治疗剂有关， 内含子剪接反应。