Ribonuclease (RNase)P catalyzes the hydrolysis of pre-tRNA with a mature 5' end. It is unique in that it is comprised of both a protein subunit and a ribonucleotide subunit, and intriguingly, the RNA subunit is responsible for catalysis. The discovery of catalytic RNA surprised the scientific community because it was initially thought that only protein molecules were diverse enough to catalyze reactions in vivo. Thus, the structural characterization of catalytic RNA molecules may provide some insight as to why nature has chosen certain chemical reactions to be catalyzed by RNA. Though several models have been proposed to account for RNase P function, a detailed molecular understanding of RNase P structure-function relationships are hindered by the lack of its three- dimensional structure and an overall limited knowledge of RNA structure.
The specific aim of this proposed research entails determining the three- dimensional structure of the M1 RNA subunit of RNase P through X-ray crystallographic methods, and involves: (1) preparation and purification of M1 RNA and its pre-tRNA substrate, (2) optimization of M1 RNA crystals and growth of M1 RNA/pre-tRNA complex crystals, (3) collection of X-ray data from these crystals, (4) phasing initial electron density maps using heavy metal derivatives, and (5) building and refining the models of M1 RNA and M1 RNA/pre-tRNA complex. The expected results will provide valuable structural information regarding the mechanisms of catalysis and substrate association utilized by RNase P. Moreover, these results will add to the currently limited understanding of RNA macromolecular structural motifs.
