(1) Determine the structure of nucleolin RBD12 in complex with a natural pre, rRNA substrate and investigate the contributions of the individual RNA binding domains and linker to recognition, sequence specific binding, and stability. (2) Determine the structure of the complex of dsRBD of yeast RNAse tll with RNA stem-loop substrates. Rntl p, the yeast RNAse III, is involved in processing of a variety of snRNAs and snoRNAs, as well as rRNA. The structure of the complex of the dsRBD of Rntl p with the snR47 substrate should reveal how this domain positions the protein for specific cleavage at a site 14,16 nt away from a conserved AGNN tetraloop. (3) Determine the structure of H/ACA snoRNA with and without rRNA substrate. The H/ACAsnoRNPs are involved in pseudouridylation of rRNA and act as """"""""guides"""""""" to position the substrate in correct position to interact with the pseudouridine synthase, Cbf5 in yeast. They have conserved hairpin, hinge-hairpin-tail secondary structure. We will determine the structure of the 3' hairpin of U65 in the absence and presence of its rRNAsubstrate and study other H/ACA snoRNAs. (4) Determine the structural features of proteins Nop1, Nhp2, and conserved central domain of Garl. tn addition to the pseudouridyine synthase, these three small proteins interact with the H/ACA snoRNA to form the core H/ACA snoRNP, we will determine if they have a stable folded structure for all or part of the protein and if so determine their structures in solution. (5) Investigate the interactions of the H/ACA snoRNP proteins with the H/ACA snoRNA. The interactions of the H/ACA snoRNP proteins with the H/ACA snoRNA will be investigated both individually and in combination. Binding sites for the proteins which interact directly with the RNA will be determined, and the individual RNA-protein complexes will be studied by NMR and/or X-ray crystallography. The long term objective is this work is to provide a structural basis for understanding some of the RNA-RNA and RNA-protein interactions that take place during eukaryotic ribosome processing. These studies should also provide fundamental insights into RNA folding and tertiary interactions and recognition of NRA by diverse RNA binding proteins.
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