Studies on Methanocaldococcus jannaschii RNase P reveal insights into the roles of RNA and protein cofactors in RNase P catalysis

Abstract

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.

Interactions of a Pop5/Rpp1 heterodimer with the catalytic domain of RNase MRP

“…The formation of a complex between Pop5 and Rpp1 is consistent with the available results of two-hybrid studies and pull-down experiments performed for yeast RNases MRP/P (HouserScott et al 2002;Aspinall et al 2007) as well as with the results obtained for archaeal homologs of Pop5 and Rpp1 (Tsai et al 2006;Pulukkunat and Gopalan 2008;Honda et al 2010).…”

Ribosomal protein L7Ae is a subunit of archaeal RNase P

“…3A; see SI Text). Our previous in vitro reconstitution studies of Mja and Pfu RNase P revealed that the functional units are not individual proteins but are binary RPP complexes (POP5•RPP30 and RPP21•RPP29) (25,26). This observation led us to purify these RPP pairs as complexes after their cooverexpression in Eco; moreover, RNase P assembled from Pfu RPR and RPPs, purified either individually or as binary complexes, exhibits comparable k cat and K m values (29).…”

“…1) (24). Type M RPRs possess a smaller, atypical L15 and lack P16, P6, and P8; these changes might adversely impact substrate binding, an expectation based on findings from the related bacterial RPR (24,25). Interestingly, the inclusion of L7Ae lowers the K m to 0.044 μM for pre-tRNA Tyr processing by Mma RNase P. Clearly, RPPs (including L7Ae) somehow remedy the substrate-binding defects of the Mma RPR.…”

“…This region is the high-affinity binding site of L7Ae in Pho RPR, and its absence in type M archaeal and all eukaryal RPRs suggests that a universal role for L7Ae and its homologs in RNase P catalysis must involve another RPR region. Therefore, we used a type M archaeal RNase P to investigate if L7Ae is indeed part of the holoenzyme and to understand how it influences catalysis.Using purified recombinant subunits, we and others have reconstituted type A and M RNase P from different thermophilic archaeal variants [Mth, Pho, Pyrococcus furiosus (Pfu) and Methanocaldococcus jannaschii (Mja)] (13,15,25,26). Because we expected studies on a mesophilic type M variant to yield results that are also applicable to eukaryal RNase P, we decided to focus on RNase P from Methanococcus maripaludis (Mma); this choice Author contributions: I-M.C…”

“…Using purified recombinant subunits, we and others have reconstituted type A and M RNase P from different thermophilic archaeal variants [Mth, Pho, Pyrococcus furiosus (Pfu) and Methanocaldococcus jannaschii (Mja)] (13,15,25,26). Because we expected studies on a mesophilic type M variant to yield results that are also applicable to eukaryal RNase P, we decided to focus on RNase P from Methanococcus maripaludis (Mma); this choice was also inspired in part by the fact that transformation and homologous recombination are possible for Mma (27,28).…”