EXON LIGATION--THE SPLICEOSOME AND GROUP 11 INTRONS

通过RNA剪接去除内含子是 几乎所有人类基因的表达。许多新的成绩单是 受制于替代剪接，这提供了一种制造更多 而不是来自单一基因的一种蛋白质。这种可选剪接可以 无论是发育控制还是组织特异性控制。不管是不是 RNA的剪接也受到监管：控制水平 在细胞质中剪接和非剪接的病毒RNA对 艾滋病毒的复制。因此，对剪接过程有详细的了解 对于更好地理解 基因表达，以及生物体发育、肿瘤发生和 逆转录病毒感染的进展。 因为剪接体的加工和基团的自我催化切除 II内含子通过相同的两步途径发生，两种内含子类型是 被认为在进化上有亲缘关系。因此，剪接体已经很长时间了 被怀疑是一种RNA催化剂。然而，精准的构成 催化中心(S)仍然难以捉摸，主要是因为剪接体 直接与剪接位点磷酸盐相互作用的组件具有 还没有确定身份。也不知道活跃的站点在多大程度上 对于剪接的两个步骤重叠，尽管越来越多的证据支持 这两个步骤之间进行了某种结构上的重新安排。更少 已知其催化机理和活性中心结构。 第二组内含子。因此，共同的两步走路径是否导致了 从这些内含子类型的发散或收敛进化中 值得商榷。此外，有哪些机械上的相似之处，如果有的话， 在上述内含子和L组自剪接类的内含子之间 是完全未知的。 这个实验室的一个长期目标是阐明催化剂 剪接体所利用的机制和活性部位机械 第二组内含子催化内含子切除。这项提案将重点放在 主要是外显子连接步骤。将在以下方面使用的并行方法 这两个体系都包括：(L)全面发展的动力 外显子连接的框架，使用其中3‘剪接位点的实验 添加在反式中；(2)使用化学修饰的3‘剪接位点RNA来 确定外显子连接的准确底物结构要求， 以及这些结构如何有助于剪接位点识别和 催化；以及(3)光和亲和交联剂的引入 用于识别活性部位成分的试剂，这些成分与 3‘剪接位点处的磷酸二酯骨架。加在一起，这些 实验应该允许直接比较活性中心的结构，3‘ 剪接体的剪接位点识别及其催化机制 组II和组I均为自剪接内含子。

The removal of introns by RNA splicing is an essential step in the expression of almost all human genes. Many nascent transcripts are subject to alternative splicing, which provides a means for making more than one protein from a single gene. Such alternative splicing can be either developmentally or tissue-specifically controlled. Whether or not an RNA is spliced is also subject to regulation: controlling the level of spliced and unspliced viral RNA in the cytoplasm is critical to the replication of HIV. Thus a detailed knowledge of the splicing process will be essential for better understanding of the basic mechanisms of gene expression, as well as organismal development, oncogenesis and the progression of retroviral infection. Because spliceosomal processing and the self-catalyzed excision of group II introns occur via the same two-step pathway, the two intron types are thought to be evolutionarily related. Therefore, the spliceosome has long been suspected to be an RNA catalyst. However, the precise makeup of the catalytic center(s) has remained elusive, primarily because spliceosomal components that interact directly with the splice site phosphates have yet to be identified. Neither is it known to what extent the active sites for the two steps of splicing overlap, although growing evidence supports some sort of structural rearrangement between the two steps. Even less is known about the catalytic mechanisms and active site structure of group II introns. Thus, whether the common two-step pathway has resulted from divergent or convergent evolution of these intron types remains debatable. Additionally, what mechanistic similarities exist, if any, between the above introns and introns of the group l self-splicing class are totally unknown. A long-term goal of this laboratory is to elucidate the catalytic mechanisms and active site machinery utilized by the spliceosome and group II introns to catalyze intron excision. This proposal focuses primarily on the exon ligation step. Parallel approaches to be used in both systems include: (l) development of a comprehensive kinetic framework for exon ligation using an assay in which the 3' splice site is added in trans; (2) use of chemically modified 3' splice site RNAs to determine the exact substrate structural requirements for exon ligation, and how these structures contribute to splice site recognition and catalysis; and (3) incorporation of photo- and affinity-crosslinking reagents to identify active site components closely juxtaposed to the phosphodiester backbone at the 3' splice site. Together, these experiments should allow direct comparison of active site structure, 3' splice site recognition and mechanisms of catalysis by the spliceosome and both group II and group I self-splicing introns.