Composition and characterization of nuclear ribonuclease P from Saccharomyces cerevisiae.

Ribonuclease P (RNase P) is the enzyme responsible for 5' processing of precursor tRNA (pre-tRNA) molecules. The holoenzyme is composed of both RNA and protein subunits. Bacterial RNase P has been studied extensively and the RNA subunits have been shown to be catalytic. The single RNase P protein subunit from bacterial enzymes is required for function in vivo. The eukaryotic nuclear RNase P RNA subunit has been identified and analyzed, but purification of the protein component of the RNase P holoenzyme has proven difficult due to its lability and low abundance. Characterization of RNase P was initiated with analysis of an RNA subunit mutation. The yeast strain harboring the RNA mutation displayed defects in precursor rRNA (pre-rRNA) as well as pre-tRNA processing. This analysis of a known RNase P subunit confirmed a close evolutionary and functional relationship between RNase P and RNase MRP, an endoribonuclease that is involved in pre-rRNA processing. To address the question of what proteins were necessary for RNase P function, the RNase P holoenzyme was purified using ion-exchange chromatography and density gradient sedimentation. Identification of a large complex of at least nine proteins in association with RNase P activity was obtained by mass spectrometry methods. Proteins found among these include four candidate RNase P subunits identified by genetic methods intended to find RNase MRP components. Five other proteins were products of hypothetical open reading frames in the S. cerevisiae genome. Employing genetic methods available in yeast, future efforts will focus on determining the role of these proteins in pre-tRNA processing, pre-rRNA processing, and other possible cellular functions.PhDBiological SciencesCellular biologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132046/2/9938579.pd