The universal enzyme ribonuclease P (RNase P) carries out maturation of transfer RNAs (tRNAs) by cleavage of precursor-tRNAs. RNase P is an unusual enzyme in that it is a ribonucleoprotein (RNP) and its catalytic component is comprised of RNA instead of the usual protein. RNA-RNA and RNA-protein interactions are critical to cellular activities, so RNase P offers a specific example of molecular structure and function with broader implications to other cellular functions. The overall goal of this proposal is to determine the structure of bacterial RNase P RNA (~400 nucleotides) and its complexes with RNase P protein (~120 amino acids) and tRNA substrate (~80 nt). Knowledge of these structures is critical to progress in RNase P research. This laboratory has long worked toward this goal, with considerable success, first with biochemical methods and most recently with determination of the crystal structure of the RNase P RNA from Bacillus stearothermophilus at 3.3? and a designed, rapidly crystallizing RNA at 3.6?. However, parts of the crystal structures are distorted or not resolved, and the specific structures of the holoenzyme and ternary complex with tRNA are not known. Even the location of the active site is questionable. At the current stage of resolution, mechanistically relevant properties such as metals, H-bonded networks and waters are not discerned. Moreover, little is known about the solution structure, an important consideration because of potential distortion and disorder in any crystal structures. To address these and other questions we propose to continue crystallographic studies underway to determine molecular structures pertinent to RNase P. To complement, test and refine structure models, small angle X-ray scattering (SAXS) is being used to determine solution structures for comparisons. SAXS results coupled with available crystal structure information will permit modeling the global solution envelope to ~10?, Crystallographic efforts include ongoing analysis of recent data sets, and screens for crystals of RNase P RNA and complexes with better diffraction quality than so far obtained. This is being approached using phylogenetically diverse native RNAs and components, and mutant derivatives thereof. For SAXS studies, we have partnered with experts at Scripps Research Institute and Lawrence Berkeley National Laboratory, and early results show this is a fruitful approach.
The proposed program will further our understanding of the unusual RNA enzyme RNase P. RNase P is universally distributed, required for all cellular growth and development, and potentially is a target for organism-specific antibiotic therapies.
| Kazantsev, Alexei V; Rambo, Robert P; Karimpour, Sina et al. (2011) Solution structure of RNase P RNA. RNA 17:1159-71 |
| Marquez, Steven M; Chen, Julian L; Evans, Donald et al. (2006) Structure and function of eukaryotic Ribonuclease P RNA. Mol Cell 24:445-56 |
| Frank, D N; Harris, M E; Pace, N R (1994) Rational design of self-cleaving pre-tRNA-ribonuclease P RNA conjugates. Biochemistry 33:10800-8 |
| LaGrandeur, T E; Huttenhofer, A; Noller, H F et al. (1994) Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA. EMBO J 13:3945-52 |
| Harris, M E; Nolan, J M; Malhotra, A et al. (1994) Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA. EMBO J 13:3953-63 |
| Brown, J W; Haas, E S; Gilbert, D G et al. (1994) The Ribonuclease P database. Nucleic Acids Res 22:3660-2 |
| Oh, B K; Pace, N R (1994) Interaction of the 3'-end of tRNA with ribonuclease P RNA. Nucleic Acids Res 22:4087-94 |
| Waugh, D S; Pace, N R (1993) Gap-scan deletion analysis of Bacillus subtilis RNase P RNA. FASEB J 7:188-95 |
| Brown, J W; Haas, E S; Pace, N R (1993) Characterization of ribonuclease P RNAs from thermophilic bacteria. Nucleic Acids Res 21:671-9 |
| Zito, K; Huttenhofer, A; Pace, N R (1993) Lead-catalyzed cleavage of ribonuclease P RNA as a probe for integrity of tertiary structure. Nucleic Acids Res 21:5916-20 |
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