RNA 97 (the RNA Society), May 27 - June 1 1997, Banff, Canada

Characterization of the catalytically inactive archaeal RNase P RNA

James Pannucci and James Brown, North Carolina State University Raleigh, NC 27695

RNase P is a ribonucleoprotein enzyme responsible for the 5' maturational cleavage of pre-tRNA. In Bacteria, the RNA subunit is responsible for catalysis, and under certain in vitro conditions, the RNase P RNA can function without the protein subunit. Much of the research concerning the RNase P RNA subunit has studied how structure relates to function in catalytically active bacterial RNA molecules. Like all bacterial species, the Archaea require RNase P to mature the 5' ends of pre-tRNAs, but under every in vitro condition tested so far, the archaeal RNase P RNA subunit by itself is not catalytically active. In an attempt to understand why the archaeal RNase P RNA is not active in vitro, despite its structural similarity to the bacterial RNase P RNA, each step of the archaeal RNase P catalytic cycle (substrate binding, catalysis, and product release) is being analyzed.

Data from mobility shift binding assays and UV crosslinking suggest that the archaeal RNase P RNA is unable to bind pre or mature tRNA. In addition, unimolecular enzyme/substrate constructs failed to cleave the pre-tRNA 5' leader sequence in cis. The RNA appears unable to perform either substrate binding or catalysis, and it seems likely that inactivity is a global structural problem. The results of these experiments, along with analysis of the archaeal RNase P RNA global structure and chimeric bacterial/archaeal RNAs, should provide further insight into the structural and functional characteristics of the RNase P RNA.