This project's objective is to apply a new biochemical method, directed hydroxyl radical mapping, to identify the RNA target of the spliceosomal DEAD-box protein Prp5p of the yeast Saccharomyces cerevisiae. Prp5p is one of many DEXD/H helicases that use energy from hydrolyzing nucleotides to alter nucleic acid structure. DEXD/H proteins share a common helicase core structure that consists of two domains with the motifs lining an inter-domain cleft. The nucleotide and magnesium ion reside at the bottom of the cleft. The target nucleic acid substrate may lie across the upper part of the cleft. Despite this structural information, the targets of the RNA helicases (including the ten spliceosomal, DEXD/H-box proteins) have yet to be identified. Preliminary data indicate that Prp5p physically interacts with U2 snRNP, its posited target, as well as pre-mRNA and even perhaps U1 snRNP. The directed hydroxyl radical mapping method will be used to discover which RNA binds to Prp5p's helicase core in the spliceosome. This in vitro method uses hydroxyl radicals generated by a Fe(II) group tethered to a protein at a specific position via the linker BABE and the sulfhydryl group of a cysteine (C). The radicals cleave any nucleic acid within 40 Angstroms. For identifying the target of Prp5p, in vitro mutagenesis will be used to create a C-less form of recombinant Prp5 protein. A single C will then be introduced at various positions within the helicase core and BABE-Fe covalently attached. Recombinant, derivatized Prp5p will then be introduced into the spliceosome in an in vitro splicing reaction. Directed hydroxyl radical cleavage of any RNA interacting with Prp5p in the spliceosome will be induced by the addition of hydrogen peroxide to the splicing reaction and any cleaved RNAs will be detected by biochemical assays. There will be several benefits resulting from this project. Because of the common structure of DEXD/H proteins, this method will be readily applicable to many other DEXD/H proteins, including the other spliceosomal DEXD/H box proteins. Furthermore, the results of this project will contribute to understanding several properties of biomolecular machines including the mechanism for converting energy from an enzymatic reaction to the work of unwinding nucleic acids. This research project will involve students at an EPSCoR and designated minority institution.
