Ribonuclease P (RNase P) is a ubiquitous endoribonuclease that cleaves precursor tRNAs to generate mature 5 termini. Although RNase P from all kingdoms of life have been found to have essential RNA subunits, the number and size of the protein subunits ranges from one small protein in bacteria to at least nine proteins of up to 100 kDa. In Saccharomyces cerevisiae nuclear RNase P, the enzyme is composed of ten subunits: a single RNA and nine essential proteins. The spatial organization of these components within the enzyme is not yet understood. In this study we examine the likely binary protein-protein and protein-RNA subunit interactions by using directed two-and three-hybrid tests in yeast. Only two protein subunits, Pop1p and Pop4p, specifically bind the RNA subunit. Pop4p also interacted with seven of the other eight protein subunits. The remaining protein subunits all showed one or more specific proteinprotein interactions with the other integral protein subunits. Of particular interest was the behavior of Rpr2p, the only protein subunit found in RNase P but not in the closely related enzyme, RNase MRP. Rpr2p interacts strongly with itself as well as with Pop4p. Similar interactions with self and Pop4p were also detected for Snm1p, the only unique protein subunit so far identified in RNase MRP. This observation is consistent with Snm1p and Rpr2p serving analogous functions in the two enzymes. This study provides a low-resolution map of the multisubunit architecture of the ribonucleoprotein enzyme, nuclear RNase P from S. cerevisiae. Ribonuclease P (RNase P) is an essential endoribonuclease that acts early in tRNA biogenesis to remove the 5Ј leader sequences of precursor tRNAs (pretRNAs) (1-3). The enzyme has been identified in every organism tested, in all kingdoms of life. In most cases, the enzyme is composed of a single RNA subunit and one or more protein subunits (1, 4). The RNA subunit forms the catalytic core of the enzyme, and the bacterial and some archaeal RNA subunits alone are catalytic in vitro (5-7). In contrast, no eukaryotic RNase P RNA subunits have been shown to be catalytic in the absence of protein. In bacteria, RNase P is composed of a catalytic RNA subunit and a single small protein subunit. Studies on the bacterial RNase P protein suggest that the protein plays a role in substrate recognition (8)(9)(10)(11). Recent data show that at least one form of archaeal RNase P consists of four or more proteins and a single RNA subunit (12). Moreover, the identified archaeal proteins appear to be homologs of the eukaryotic RNase P proteins and not the bacterial proteins (T. A. Hall and J. W. Brown, personal communication).Eukaryotic nuclear RNase P contains an RNA subunit similar in size to its bacterial and archaeal counterparts, containing all of the most conserved ''critical regions'' from the bacterial consensus structure (13). However, the protein content is far more complex and is absolutely required for activity. Human nuclear RNase P appears to contain at least ten proteins (14-19). At le...

Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and protein subunits. The RNA subunits of each enzyme share substantial secondary structural features, and most of the protein subunits are shared between the two. One of the conserved RNA subdomains, designated P3, has previously been shown to be required for nucleolar localization. Phylogenetic sequence analysis suggests that the P3 domain interacts with one of the proteins common to RNase P and RNase MRP, a conclusion strengthened by an earlier observation that the essential domain can be interchanged between the two enzymes. To examine possible functions of the P3 domain, four conserved nucleotides in the P3 domain of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a library of all possible sequence combinations at those positions. Selection of functional genes in vivo identified permissible variations, and viable clones that caused yeast to exhibit conditional growth phenotypes were tested for defects in RNase P RNA and tRNA biosynthesis. Under nonpermissive conditions, the mutants had reduced maturation of the RPR1 RNA precursor, an expected phenotype in cases where RNase P holoenzyme assembly is defective. This loss of RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation characteristic of RNase P defects. To test whether mutations at the conserved positions inhibited interactions with a particular protein, specific binding of the individual protein subunits to the RNA subunit was tested in yeast using the threehybrid system. Pop1p, the largest subunit shared by RNases P and MRP, bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was eliminated by mutations at the conserved P3 residues. These results indicate that Pop1p interacts with the P3 domain common to RNases P and MRP, and that this interaction is critical in the maturation of RNase P holoenzyme.