STRUCTURE AND FUNCTION OF NUCLEIC ACIDS

描述（来自应用程序）：内含子中断了许多 真核基因它们通过mRNA剪接被去除， 磷酰基转移反应在一个复杂的和动态的分子机器， 剪接体tRNA基因也被内含子中断，但在这里， 剪接是由三种酶蛋白催化的， ATP水解。我们一直关心的是确定 酵母中的核前mRNA剪接和tRNA剪接。 在过去的资助期间，我们发现酵母tRNA内切酶， 催化tRNA剪接的第一步是α-β-γ-δ 四聚体这两个催化亚基与tRNA的剪接有关 来自古细菌的核酸内切酶。这使我们确定了 两种古细菌核酸内切酶，一种四聚体，另一种二聚体。这些结构 提出了催化和底物识别机制的模型， 古细菌和真核生物tRNA内切酶。我们建议测试这些模型， 来确定古细菌-基质复合体的结构 为了理解mRNA剪接，我们必须解决两个相互关联的问题： 剪接体的组装以及剪接是如何被催化的。我们最近的进展 实验室和现场的研究表明，我们正处于一个重大的 将这些办法结合起来，形成一套互动的具体目标： 1.在纯化酵母snRNP中，我们发现了五肽snRNP。我们提出 来扩大这个粒子的纯化规模， 组件，并使用它来建立一个系统的spliceosome组装。 2.我们将继续获得和表征RNA-RNA和RNA-蛋白质 剪接体内的交联。交联可以用作聚合物的标记物。 剪接体组装步骤。 3.我们将设计和合成RNA分子， RNA的二级和三级结构。 4.一组依赖RNA的ATP酶或解旋酶被认为在 在剪接体组装中的作用。在过去一个月，我们完成了 这个家族的一个成员的晶体结构，一种来自 古细菌H. jannaschii。我们将进一步表征这种酶， 我们将继续努力了解两种剪接体ATP酶的作用， Prp 5和Prp 22。

DESCRIPTION (from the application):Introns interrupt the continuity of many eukaryotic genes. Their removal by mRNA splicing takes place via two phosphoryltransfer reactions in a complex and dynamic molecular machine called the spliceosome. tRNA gene are also interrupted by introns, but here the splicing is catalyzed by three enzymatic proteins with an intrinsic requirement for ATP hydrolysis. We have been concerned with determining the mechanisms of both nuclear pre mRNA splicing and tRNA splicing in yeast. During the past grant period we have found that the yeast tRNA endonuclease, which catalyzes the first step in tRNA splicing is an alpha-beta-gamma-delta tetramer. The two catalytic subunits are related to the tRNA splicing endonucleases from archaebacteria. This led us to determine the structure of two archaeal endonucleases, one tetrameric, the other dimeric. These structures suggest models for the mechanisms of catalysis and substrate recognition in archaeal and eukaryotic tRNA endonucleases. We propose to test these models and to determine the structure of an archaeal-substrate complex. To understand mRNA splicing we must solve two interrelated questions: how is the spliceosome assembled and how is splicing catalyzed. Recent progress in our laboratory and in the field suggests that we are on the eve of significant coalescence of these approaches leading to an interactive set of specific aims: 1. In purifying the yeast snRNPs we have discovered a penta snRNP. We propose to scale up the purification of this particle, to catalog its protein components and to use it to establish a system for spliceosome assembly. 2. We will continue to obtain and characterize RNA-RNA and RNA-protein crosslinks within the spliceosome. Crosslinks can serve as markers for the steps of spliceosome assembly. 3. We will design and synthesize RNA molecules which embody current models of secondary and tertiary RNA structure in the active spliceosome. 4. A set of RNA dependent ATPases or helicases are thought to play a crucial role in spliceosome assembly. During the past month we have completed the crystal structure of a member of this family, a "DEAD ' protein from the archaebacterium, H. jannaschii. We will further characterize this enzyme and we will continue our efforts to understand the role of two spliceosomal ATPases, Prp5 and Prp22.