Characterization of the RNA-binding properties of Pop3p, an essential ribonuclease P protein component from Saccharomyces cerevisiae

Studies of RNA-protein complexes over the last two decades have dramatically changed the way scientists view enzyme catalysis. Until recently, it was widely accepted that proteins carry out all enzyme-catalyzed reactions inside cells. It is now known, however, that RNA can function as both substrate and enzyme. The RNA subunit of the bacterial enzyme ribonuclease P (RNase P) was the first RNA shown to possess intrinsic catalytic activity in the absence of protein. The RNase P holoenzyme is composed of both protein and RNA in all three kingdoms of life, although the compositions of the holoenzymes differ significantly. The bacterial holoenzyme contains a single catalytic RNA subunit and a single auxiliary protein subunit. In contrast, the holoenzyme from the eukaryotic microorganism Saccharomyces cerevisiae has been shown to contain as many as nine proteins and a single RNA species whose catalytic functions are lacking in vitro. The eukaryotic proteins may provide essential functions that are otherwise lacking in a RNA-alone system.;The goal of my project was to identify and to characterize RNA-protein interactions for RNase P from S. cerevisiae using reconstituted RNA-protein subcomplexes. The results reported herein suggest that protein(s) provide substrate-binding functions for eukaryotic RNase P holoenzymes. Pop3p was shown to possess novel RNA-binding properties that included a preference for single-stranded RNA molecules and the ability to bind RNA with very high affinity. Further, Pop3p was shown to recognize a region on a pre-tRNA substrate that encompasses the 5' leader sequence and spans the natural RNase P processing site. Together, these studies support a role for eukaryotic RNase P proteins as major contributor to active site architecture, and suggest a functional hand-off from a specific RNA domain to a protein component with regard to substrate-binding responsibilities for holoenzyme function.