No abstract

RNase P, which catalyzes the magnesium-dependent 5-end maturation of tRNAs in all three domains of life, is composed of one essential RNA and a varying number of protein subunits depending on the source: at least one in bacteria, four in archaea, and nine in eukarya. To address why multiple protein subunits are needed for archaeal͞eukaryal RNase P catalysis, in contrast to their bacterial relative, in vitro reconstitution of these holoenzymes is a prerequisite. Using recombinant subunits, we have reconstituted in vitro the RNase P holoenzyme from the thermophilic archaeon Pyroccocus furiosus (Pfu) and furthered our understanding regarding its functional organization and assembly pathway(s). Whereas Pfu RNase P RNA (RPR) alone is capable of multiple turnover, addition of all four RNase P protein (Rpp) subunits to Pfu RPR results in a 25-fold increase in its k cat and a 170-fold decrease in Km. In fact, even in the presence of only one of two specific pairs of Rpps, the RPR displays activity at lower substrate and magnesium concentrations. Moreover, a pared-down, mini-Pfu RNase P was identified with an RPR deletion mutant. Results from our kinetic and footprinting studies on Pfu RNase P, together with insights from recent structures of bacterial RPRs, provide a framework for appreciating the role of multiple Rpps in archaeal RNase P.archaeal RNase P ͉ in vitro reconstitution ͉ precursor tRNA processing R ibonuclease P (RNase P) is an ancient and essential endoribonuclease that catalyzes the 5Ј-end maturation of tRNAs in all three domains of life (1-7). Whereas RNase P in all living organisms contains one essential RNA subunit, the number of protein cofactors͞subunits varies: at least one in bacterial (8), four in archaeal (9), and nine in eukaryal (nuclear) RNase P (10, 11). The basis for this variation, which has implications for macromolecular evolution, is unclear.The bacterial RNase P RNA (RPR) alone is catalytically active under in vitro conditions of high ionic strength in the presence of a divalent ion such as Mg 2ϩ (1); however, the protein cofactor is essential for RNase P function in vivo because of its pleiotropic effects on RNA structure, substrate recognition, affinity for Mg 2ϩ , and precursor tRNA (ptRNA) cleavage (12-19). Although phylogenetic sequence analysis revealed that archaeal and eukaryal RPRs likely retain the same catalytic core as the bacterial ribozyme, many archaeal and all eukaryal RPRs seem to be incapable of supporting catalysis in the absence of their protein cofactors (6,7,20,21). Genetic and biochemical studies established the association of yeast͞human nuclear RNase P activity with at least nine protein subunits (seven of which are homologs) (10, 11). RNase P activity could be immunoprecipitated from a partially purified Methanothermobacter thermoautotrophicus (Mth) RNase P preparation by using polyclonal antisera generated against four Mth polypeptides that exhibit sequence homology to four of the seven conserved yeast͞human RNase P proteins (Rpps; ref. 9). None of the archaeal...

Ribonuclease P (RNase P), a ribonucleoprotein (RNP) complex required for tRNA maturation, comprises one essential RNA (RPR) and protein subunits (RPPs) numbering one in bacteria, and at least four in archaea and nine in eukarya. While the bacterial RPR is catalytically active in vitro, only select euryarchaeal and eukaryal RPRs are weakly active despite secondary structure similarity and conservation of nucleotide identity in their putative catalytic core. Such a decreased archaeal/eukaryal RPR function might imply that their cognate RPPs provide the functional groups that make up the active site. However, substrate-binding defects might mask the ability of some of these RPRs, such as that from the archaeon Methanocaldococcus jannaschii (Mja), to catalyze precursor tRNA (ptRNA) processing. To test this hypothesis, we constructed a ptRNA-Mja RPR conjugate and found that indeed it self-cleaves efficiently (kobs, 0.15 min−1 at pH 5.5 and 55°C). Moreover, one pair of Mja RPPs (POP5-RPP30) enhanced kobs for the RPR-catalyzed self-processing by ∼100-fold while the other pair (RPP21-RPP29) had no effect; both binary RPP complexes significantly reduced the monovalent and divalent ionic requirement. Our results suggest a common RNA-mediated catalytic mechanism in all RNase P and help uncover parallels in RNase P catalysis hidden by plurality in its subunit make-up.