Abstract
Ribonuclease P (RNase P) catalyzes the 5'-end maturation of transfer RNA molecules. 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 protein 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 products. The recognition sequence for the H6Pop3p-substrate interaction in vitro was mapped to a 39-nucleotide long sequence that extends from position -21 to +18 surrounding the natural processing site in pre-tRNA substrates. H6Pop3p binds a variety of RNA molecules with high affinity (K(d) = 16-25 nm) and displays a preference for single-stranded RNAs. Removal or modification of basic C-terminal residues attenuates the RNA-binding properties displayed by the protein specifically for a pre-tRNA substrate. These studies support the model that eukaryotic RNase P proteins bind simultaneously to the RNA subunit and RNA substrate.
Highlights
We have previously reported that proteins constitute significant components of the active site architecture for the eukaryotic Ribonuclease P (RNase P) holoenzyme [7]
The lack of a demonstrable catalytic activity for eukaryotic RNase P RNAs suggests a more critical role for protein subunits in eukaryotes than the protein subunit provides in the bacterial holoenzyme
The lack of catalytic activity in eukaryotic RNase P RNA-alone reactions and the apparent abundance of protein subunits in the eukaryotic holoenzyme that lie in the vicinity of the pre-transfer RNA (tRNA) substrate led to our original proposal that the eukaryotic RNase P holoenzyme possesses a proteinrich active site [7]
Summary
Materials—All buffer solutions were prepared with sterile water that was initially deionized using a Millipore MilliQ water purification system. End-labeled RNAs were prepared for use in the filter retention experiments. To prepare 3Ј-end-labeled pretRNA, ϳ100 pmol of pre-tRNA was incubated with 6.7 pmol of [5Ј32P]cytidine 3Ј,5Ј-bisphosphate in the presence of 10 units of T4 RNA ligase RNA ligase buffer (50 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 5 mM dithiothreitol, 1 mM ATP) in a 10-l reaction volume for 2 h at 37 °C. The nitrocellulose filters were immediately washed in 2 ml of TE/NaCl buffer (0.25 M NaCl/0.01 M Tris-HCl (pH 8.0)/0.001 M Na2EDTA) containing 2% SDS for 2 h This mixture was extracted with the addition of an equal volume of phenol for 2 h. The sequencing standards were prepared by subjecting the end-labeled, full-length RNAs to limited alkaline hydrolysis or partial RNase T1 digestion
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