RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein.We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPRI (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPRI 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPRI RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPRI transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPRI-disrupted haploids with one variant of RPRI gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPRI RNA is an essential component of this enzyme.RNase P from both procaryotic and eucaryotic sources is an endonuclease that cleaves pre-tRNA substrates to yield mature 5' termini. In procaryotes, both the RNA and protein components of the ribonucleoprotein enzymes are required for in vivo activity (15,27,29,33,43,49,57,58), but the RNA component alone is capable of efficiently catalyzing the correct reaction under some conditions in vitro (3,18,21,22,52). Extensive phylogenetic sequence comparisons of these RNAs (30), combined with folding energy calculations and cleavage sensitivity studies (6, 19, 51, 52), suggest a conserved, highly ordered secondary structure. Most if not all of the key contacts with the substrates depend on this structure, with the protein contributing to efficiency through secondary effects such as charge shielding between RNA chain phosphate backbones. The mechanism by which pretRNA substrates are recognized is not clearly understood, however, since there are no obvious regions of required Watson-Crick base pairing between the enzyme and substrate RNAs (20,36).Studies of eucaryotic RNase Ps have suggested that they also contain essential RNA subunits, although the role of the RNA components has not been firmly established. RNAs that copurify with both nuclear and organelle RNase Ps have been characterized (1,7,9,11,12,14,24,32,34,35,37,38,40,42) and shown to have sequence and structural similarities to each other and to a lesser degree to the procaryotic RNAs (5, 37). The identification of the eucaryotic RNAs as essential RNase P subunits has not been rigorously confirmed, however, since there are no reports of the eucaryotic RNAs having enzyme activity in the absence ...

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.