STRUCTURE-SENSITIVE RNA FOOTPRINTING OF YEAST NUCLEAR RIBONUCLEASE-P

Several enzymatic and chemical reagents were used to probe the seconda ry structure of Saccharomyces cerevisiae nuclear RNase P RNA in the pr esence and absence of its protein components. Double-stranded regions were detected with RNase V1 and single-stranded regions with RNase ONE (Escherichia coil RNase I). Nucleotides not paired at Watson-Crick po sitions were monitored with dimethyl sulfate, kethoxal, and hexyl-3-[2 -(N-methylmorpholinio)ethyl]carbodiimide p-toluenesulfonate. The resul ts supported most aspects of the previously proposed, phylogenetically -derived RNA secondary structure, although minor refinements allowed i ncorporation of both the biochemical and phylogenetic data. Digestion of the RNase P protein(s) with proteinase K gave enhanced reactivities to structure probes at selected positions, indicating regions of the RNA made inaccessible by the presence of the protein subunit(s). The r egions of RNA protected in the yeast nuclear holoenzyme were considera bly more extensive than that seen in the Escherichia coli holoenzyme, consistent with the observation that the protein moiety generally comp rises a larger percentage of the RNase P holoenzyme in eukaryotes than in eubacteria.