Novel RNA-binding properties of Pop3p support a role for eukaryotic RNase P protein subunits in substrate recognition

Ribonuclease P (RNase P) catalyzes the 5'-end maturation of transfer RNA mo lecules. Recent evidence suggests that the eukaryotic protein subunits may provide substrate-binding functions (True, H. L., and Celander, D. W. (1998 ) J. Biol Chem. 273, 7193-7196). We now report that Pop3p, an essential pro tein subunit of the holoenzyme in Saccharomyces cerevisiae, displays novel RNA-binding properties. A recombinant form of Pop3p (H6Pop3p) displays a 3- fold greater affinity for binding pre-tRNA substrates relative to tRNA prod ucts. The recognition sequence for the H6Pop3p-substrate interaction in vit ro was mapped to a 39-nucleotide long sequence that extends from position - 21 to +18 surrounding the natural processing site in pre-tRNA substrates. H 6Pop3p binds a variety of RNA molecules with high affinity (K-d = 16-25 nm) and displays a preference for single-stranded RNAs. Removal or modificatio n of basic C-terminal residues attenuates the RNA-binding properties displa yed by the protein specifically for a pre-tRNA substrate. These studies sup port the model that eukaryotic RNase P proteins bind simultaneously to the RNA subunit and RNA substrate.