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 (k(obs), 0.15 min(-1) at pH 5.5 and 55 degrees C). Moreover, one pair of Mja RPPs (POP5-RPP30) enhanced k(obs) for the RPR-catalyzed self-processing by approximately 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.

Figure 1.

An RPR hybrid comprising an S and C domain from a bacterial and an archaeal type M RPR, respectively, is catalytically active and can accurately process ptRNA^Tyr. (A) The shaded circles, ovals, and black boxes depict known interactions between the Eco RPR and a ptRNA substrate (11). Arrow indicates the site of cleavage in the ptRNA. In the RPR, the C and S domains are indicated in black and gray, respectively. Thick dotted lines indicate tertiary interactions unique to the bacterial RPR. (B and C) Secondary structures of representative archaeal type A and M RPRs, respectively. The structural elements absent in type M RPR (C) are highlighted with dotted lines. (D) Secondary structure of the bacterial/archaeal hybrid RPR (EcoS-MjaC RPR) used in this study. The S domain of bacterial RPR is indicated in gray. Universally conserved nucleotides are indicated in A–D. (E) EcoS-MjaC RPR (2.5 μM) was tested for RNase P activity by incubating at 50°C for 15 min with 5′ labeled ptRNA^Tyr (∼1 pM) in 50 mM Tris-acetate (pH 8) and different NH₄⁺ and Mg²⁺ concentrations as indicated. M represents a size marker generated by processing of ptRNA^Tyr by Eco RNase P.

Figure 2.

Design and optimization of self-cleavage conditions for an active-site tethered ES conjugate. (A) Illustration of pt^Tyr-S₃ (and S₅)-M RPR in which ptRNA^Tyr is attached to L15 of Mja RPR with either a 3- or 5-nt spacer. The C and S domains are demarcated to indicate what was deleted in pt^Tyr-S₃-ΔS M RPR. (B) Titration of monovalent and divalent cations to identify the optimal conditions for maximal self-cleavage of 5′ labeled pt^Tyr-S₅-M RPR (see text for details). R and L indicate RPR cis conjugate and the 5′ leader, respectively.

Figure 3.

Dependence of k_obs on assay pH for self-cleavage of pt^Tyr-S₃-M RPR and pt^Tyr-S₃-ΔS M RPR (A) and pt^Tyr-S₃-M RPR + POP5-RPP30 (B).

Figure 4.

Effect of Mja RPPs on the self-processing rate of pt^Tyr-S₃-M RPR and pt^Tyr-S₃-ΔS M RPR. All assays were performed in 50 mM MES (pH 5.1) at 55°C. 5′ labeled pt^Tyr-S₃-M RPR (A) or pt^Tyr-S₃-ΔS M RPR (B) was reconstituted with binary complexes of Mja RPPs at various concentrations of monovalent and divalent cations as indicated. (C) Rate of self-cleavage of the pt^Tyr-S₃-M RPR without RPPs (triangles), with RPP21-RPP29 (circles) or with POP5-RPP30 (squares); assay conditions are listed in Table 1.