Dr. Hampel will characterize a novel catalytic RNA/substrate RNA complex - the proposed "hairpin." The catalytic complex consists of a short catalytic RNA of 50 bases of satellite sequence and a substrate RNA of 14 bases of satellite sequences. The catalytic RNA cleaves the substrate at an ApG bond with a Km=0.03uM and kcat=2.1/min in the presence of 12mM MgC12, 40mM Tris pH=7.5 at 37C which, under these mild conditions, is the most active RNA catalysis known. The model will be tested by site-directed mutagenesis, structure- specific nuclease digestion, and large scale RNA chemical synthesis such that others can analyze the catalytic RNA/substrate complex by NMR and X-ray crystallography. The research area of this proposal is currently creating considerable interest, not only because of its importance in the replication of certain plant pathogens, but also because of this fundamental importance in contributing to our understanding of RNA catalysis and the RNA world. So far there appear to be three types of autolytically cleaving RNAs. The best characterized to date is the "hammerhead" structure associated with one viroid, one or more virusoids, and an RNA transcript of newt DNA. The one described here is another where a different active structure appears likely. The third is that of the hepatitis B delta factor RNA, where a third type of structure is probable.
