(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.
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