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。通过mRNA剪接移除它们是通过两个 复杂动态分子机器中的磷酸化转移反应 剪接体。TRNA基因也被内含子打断，但在这里 剪接是由三种具有内在要求的酶蛋白催化的 用于ATP水解液。我们一直关注于确定 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剪接的第一步是一个α-β-伽马-三角洲 四聚体。这两个催化亚基与trna剪接有关。 来自古细菌的内切酶。这导致我们确定了 两种古代核酸内切酶，一种是四聚体，另一种是二聚体。这些结构 提出了催化和底物识别机制的模型 古生物和真核生物的tRNA内切酶。我们建议测试这些模型并 以确定古生物-底物复合体的结构。 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催化的。我们的最新进展 实验室和实地研究表明，我们正处于意义重大的前夕 这些方法的结合导致了一套互动的具体目标： 1.在酵母SnRNP的纯化过程中，我们发现了一个五链的SnRNP。我们建议 放大这种颗粒的提纯，对其蛋白质进行分类 组件，并利用它来建立剪接体组装系统。 2.我们将继续获得和鉴定RNA-RNA和RNA-蛋白质 剪接体内的交联物。交联物可以作为标记 剪接体组装的步骤。 3.我们将设计和合成体现当前模型的RNA分子 活性剪接体中的二级和三级RNA结构。 4.一组依赖于RNA的ATPase或解旋酶被认为在 在剪接体组装中的作用。在过去的一个月里，我们完成了 这个家族的一个成员的晶体结构，一种来自 古生菌H.jannaschii。我们将进一步鉴定这种酶，我们将 我们将继续努力了解两个剪接体ATPase的作用， Prp5和Prp22。

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.