Structure/Function Analysis of Rnase P, an RNA Catalyst

Marsh, Terry

Abstract

The potential dynamic aspect of RNA molecules is underscored by the endonucleolytic activity of RNase P RNA on its substrate, precursor tRNA. RNase P RNA was the second RNA to be described with catalytic activity. We know little about the structure of this molecule or its configuration within the cell, and even less about the ubiquity of RNase P throughout the three kingdoms. This research proposal addresses these shortcomings and proposes to extend the search for biologically important small RNAs to other species and kingdoms in the following ways. 1. Continued analysis of RNase P RNA structure. One of the most reliable methods of structure analysis of nucleic acids is comparative sequence analysis. We intend to isolate and sequence RNase P RNA from several representative organisms as a means of determining secondary structure. 2. Search for RNase P in distantly related genera and kingdoms. Of particular interest to us is the nature of RNase P activity in the Archaebacteria, an ancient lineage of organisms. We plan to search for RNase P in Halobacterium volcanii, H. salinarium, and possibly Sulfolobus solfataricus. 3. Assessment of the influence of other cellular components of the activity and conformation of B. subtilis RNase P. During the isolation of RNase P protein from B. subtilis, 14 proteins were purified to near homogeneity. At lease one of these proteins was found to inhibit RNase P activity. We propose to further examine these proteins for inhibitory or stimulatory activity as well as determine their affinity, if any, for RNase P RNA and tRNA. 4. Examination of the structure of the tRNA substrate under conditions optimal for RNase P activity. It is of course the substrate that is cleaved and therefore cleavage may be critically dependent on the conformation of the tRNA substrate. We plan to investigate this possibility using spectrophotometric and enzymatic analysis of precursor and mature substrate structure under optimal RNA processing conditions. 5. We will examine the constellation of small RNAs within representative microorganisms including B. subtilis, B. brevis, Streptoccus faecium, and H. vulcanii. The RNAs will be isolated by a combination of column chromatography and gel electrophoresis, characterized by Southern analysis and sequenced when possible.