DEFINITION OF THE CATALYTIC SITE OF THE GROUP II INTRON

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。这目前是不可能的，因为我们缺乏足够的 详细了解人前信使核糖核酸内含子的作用机制 拼接。前-mRNA剪接是由剪接体催化的，剪接体 由60种不同的蛋白质和5个RNA组成。体能 剪接体的复杂性阻碍了对剪接的研究。这个 某些真菌内含子的剪接不需要剪接体 与人类前信使核糖核酸内含子相关的基因，即自剪接第二组 内含子。第二组内含子剪接的研究与研究相关 因为第二组内含子剪接发生在相同的 机理，包括两个连续的酯交换反应，使用 在剪接体旁边。综上所述，第二组自剪接内含子是一种 人前信使核糖核酸剪接研究的简单模型系统。定义 第二类内含子的催化核心将有助于定义 剪接体的催化核心。这些研究将提供基本的 与应用研究相关的信息，旨在发现小说 治疗学。 一个基本的问题是，第二组内含子和剪接体， 使用单个活性中心或两个活性中心来催化这两个 剪接的反应。调查员已经开发出一种系统， 将使他能够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裂解反应上。一 切割模拟剪接的第一个反应，另一个模拟剪接的 第二反应。如果核酶有一个活性部位，相同的 两种切割反应都需要序列。 第一个基因需要一个特殊的内含子序列，结构域5。 剪接反应。第二个是否需要域5 反应尚不清楚。缺乏信息的一个原因是 阻止第一个反应的突变也会阻止第二个反应，通过 默认情况下，因为拼接发生在两个连续的反应步骤中。至 绕过这个问题，调查员将产生两个RNA， 如果缺少结构域5的内含子能够完成第一步 拼接。这些RNA完成第二反应的能力 将会受到考验。这些研究将确定领域5是否是 第二个反应所需的。 结构域5与位于内含子另一区域的位点紧密结合。 这种交互可能形成活动站点的关键组件 催化第一个反应。结合是通过一种新的第三代 互动。结构域5的结合伙伴尚未找到。这个 这种相互作用的三维结构可以使用交叉 连接、铁-EDTA和诱变。这些研究将描述一种 新的RNA-RNA相互作用可能是人类Pre-Pre所必需的 信使核糖核酸剪接 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 与控制特定疾病的治疗剂的设计有关 内含子剪接反应。

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.