(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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM037254-17
Application #
6679411
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
1986-07-01
Project End
2007-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
17
Fiscal Year
2003
Total Cost
$358,413
Indirect Cost
Name
University of California Los Angeles
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Hartman, Elon; Wang, Zhonghua; Zhang, Qi et al. (2013) Intrinsic dynamics of an extended hydrophobic core in the S. cerevisiae RNase III dsRBD contributes to recognition of specific RNA binding sites. J Mol Biol 425:546-62
Wang, Zhonghua; Hartman, Elon; Roy, Kevin et al. (2011) Structure of a yeast RNase III dsRBD complex with a noncanonical RNA substrate provides new insights into binding specificity of dsRBDs. Structure 19:999-1010
Koo, Bon-Kyung; Park, Chin-Ju; Fernandez, Cesar F et al. (2011) Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface. J Mol Biol 411:927-42
Kim, Nak-Kyoon; Theimer, Carla A; Mitchell, James R et al. (2010) Effect of pseudouridylation on the structure and activity of the catalytically essential P6.1 hairpin in human telomerase RNA. Nucleic Acids Res 38:6746-56
Singh, Mahavir; Gonzales, Fernando A; Cascio, Duilio et al. (2009) Structure and functional studies of the CS domain of the essential H/ACA ribonucleoparticle assembly protein SHQ1. J Biol Chem 284:1906-16
Kim, Nak-Kyoon; Zhang, Qi; Zhou, Jing et al. (2008) Solution structure and dynamics of the wild-type pseudoknot of human telomerase RNA. J Mol Biol 384:1249-61
Wu, Haihong; Feigon, Juli (2007) H/ACA small nucleolar RNA pseudouridylation pockets bind substrate RNA to form three-way junctions that position the target U for modification. Proc Natl Acad Sci U S A 104:6655-60
Khanna, May; Wu, Haihong; Johansson, Carina et al. (2006) Structural study of the H/ACA snoRNP components Nop10p and the 3' hairpin of U65 snoRNA. RNA 12:40-52
Qin, Peter Z; Feigon, Juli; Hubbell, Wayne L (2005) Site-directed spin labeling studies reveal solution conformational changes in a GAAA tetraloop receptor upon Mg(2+)-dependent docking of a GAAA tetraloop. J Mol Biol 351:1-8
Wu, Haihong; Finger, L David; Feigon, Juli (2005) Structure determination of protein/RNA complexes by NMR. Methods Enzymol 394:525-45

Showing the most recent 10 out of 54 publications