Most eukaryotic genes contain introns which must be removed from pre-mRNA by the spliceosome prior to translation. The spliceosome is a mega-Dalton macromolecular machine composed of both RNA and protein components. ATP is not utilized for chemical bond formation during either lariat synthesis or exon-exon ligation by the spliceosome. Nevertheless, ATP hydrolysis is required for structural rearrangements that are essential for product formation. These structural rearrangements result in defined, stable complexes that have been isolated and studied in vitro. It is not known how transitions between complexes occur, but one hypothesis is that essential DExD/H-box proteins (Prp2,16, 22, and 43) initiate structural reorganization of the spliceosome by using the energy of ATP hydrolysis to disrupt protein/RNA or RNA/RNA interactions. Given the ~3 MDa size of the spliceosome, traditional biochemical analysis of how these enzymes facilitate splicing is difficult. In this research proposal, single molecule fluorescence (SMF) will be used as a new method to study interactions between DExD/H-box proteins and the spliceosome. Spliceosomes will be assembled on a derivitized glass surface from Prp2-deficient yeast cell extract. Due to the absence of Prp2, the spliceosomes will assemble on a surface-immobilized, fluorescently-tagged pre-mRNA substrate but will become stalled prior to lariat formation. The Prp enzymes will be expressed and purified from E. coli using published procedures and fluorescently-labeled using either a genetically-encoded GFP variant or keto-biotin. Interactions between the enzymes and the immobilized spliceosomes will be visualized using multi-wavelength SMF microscopy. These experiments will provide new insight into spliceosome catalysis not attainable with ensemble methods by unambiguously testing the proposed transient association of each DExD/H-box protein with the spliceosome and the role of ATP hydrolysis and conserved protein domains in spliceosome binding. RELEVANCE:Splicing of pre-mRNA transcripts is an essential step in gene expression, and errors in pre- mRNA splicing have been correlated with a number of cancers including infant brain tumors, breast cancers, and retinoblastomas. Understanding how the spliceosome catalyzes splicing is critical for understanding gene expression in both healthy and diseased eukaryotic cells. Elucidation of DExD/H-box protein interactions with the spliceosome using single molecule fluorescence will provide new insight into the chemical steps of splicing not possible with ensemble measurements. [unreadable] [unreadable] [unreadable]