RUI: Structrual Analysis of RNase P, An RNA Catalyst

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The dynamic capabilities of RNA have been fully realized with the discovery that RNA molecules can perform as catalysts in secondary reactions of the central dogma. The endonucleolytic activity of RNase P RNA on its substrate, precursor tRNA, was the second such activity to be described. It is proposed to continue the investigation on the structure of RNase P in order to further define the mechanism of catalysis and the intracellular structure of RNase P. Further it is proposed to extend the understanding of the phylogenetics of this RNA catalyst as well as to begin a search for other physiologically important small RNAs. The structural analysis of RNase P RNA will make use of comparative sequencing of RNase P RNAs from several microorganisms as well as chemical and enzymatic probes of structure at various temperatures and ionic conditions. The phylogenetics of RNase P will be explored by examining Deinococcus radiodurans, Micrococcus roseus, Clostridium sporogenes, Halobacter solanarium, and Thermoplasma acidophilum using a synthetic precursor tRNA as substrate. The intracellular conformation of the RNase P will be examined by the isolation of the activity under conditions that preserve macromolecular interactions and, alternatively, by asking what proteins bind to purified RNase P RNA. Other small RNAs isolated will be tested for evolutionary conservation (hence physiological importance). RNA has recently been found to catalyze certain reactions in its own maturation process. This is contrary to the previously held concept that only proteins could serve as catalysts (enzymes). It also leads to the obvious possibility that RNA molecules were the first self-replicating units of life. This proposal is to investigate the structure of these RNA particles and to determine whether they are present in the most ancient forms of life still present on the planet, the archaeacteria.