Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex

Abstract

RNase P catalyzes the Mg(2)(+)-dependent 5'-maturation of precursor tRNAs. Biochemical studies on the bacterial holoenzyme, composed of one catalytic RNase P RNA (RPR) and one RNase P protein (RPP), have helped understand the pleiotropic roles (including substrate/Mg(2+) binding) by which a protein could facilitate RNA catalysis. As a model for uncovering the functional coordination among multiple proteins that aid an RNA catalyst, we use archaeal RNase P, which comprises one catalytic RPR and at least four RPPs. Exploiting our previous finding that these archaeal RPPs function as two binary RPP complexes (POP5•RPP30 and RPP21•RPP29), we prepared recombinant RPP pairs from three archaea and established interchangeability of subunits through homologous/heterologous assemblies. Our finding that archaeal POP5•RPP30 reconstituted with bacterial and organellar RPRs suggests functional overlap of this binary complex with the bacterial RPP and highlights their shared recognition of a phylogenetically-conserved RPR catalytic core, whose minimal attributes we further defined through deletion mutagenesis. Moreover, single-turnover kinetic studies revealed that while POP5•RPP30 is solely responsible for enhancing the RPR's rate of precursor tRNA cleavage (by 60-fold), RPP21•RPP29 contributes to increased substrate affinity (by 16-fold). Collectively, these studies provide new perspectives on the functioning and evolution of an ancient, catalytic ribonucleoprotein.

Figure 1.

Secondary structure representations of (A) Eco, (B) Mth, (C) Mja and (D) Hsa RPRs (12,15). Letters indicate universally conserved nucleotides (12). Paired helices (e.g. P1, P2) are numbered consecutively from 5′ to 3′ and according to the Eco RPR nomenclature (12). Alternative secondary structure representations (69) are provided in Supplementary Figure S1.

Figure 2.

SDS–PAGE profiles depicting the purification of binary complexes of Mja RPPs: RPP21•RPP29 (top panel) and POP5•RPP30 (bottom panel). IP represents the ammonium sulfate-fractionated sample that was subjected to the SP-Sepharose column chromatography and FT denotes the flow through.

Figure 3.

Reconstitution of RNase P activity with Mja (top panel) or Mth (bottom panel) RPR and cognate RPPs purified as binary complexes (see text for details).

Figure 4.

Identification of the smallest functional RPR (yet reported) and a minimal catalytic RNP core of Mja RNase P. (A) Secondary structure models of Mja RPR and its deletion derivatives. (B) Reconstitution of RNase P activity using the two Mja RPR deletion derivatives and various combinations of Mja RPPs as indicated.

Figure 5.

Heterologous reconstitution of an RNase P holoenzyme using Eco RPR and Pfu RPPs (in the combinations indicated).

Figure 6.

Heterologous reconstitution of an RNase P holoenzyme using Ram mt RPR and archaeal (Mja) RPPs. (A) Secondary structure representation of Ram mt RPR. (B) Reconstitution of RNase P using Ram mt RPR and Mja RPPs (in the combinations indicated).

Scheme I.

Figure 7.

Effects of Mth RPPs on the single-turnover rate of Mth RPR-catalyzed pre-tRNA cleavage. The rates of product formation (k_obs) by Mth RPR with and without RPPs were determined under single-turnover conditions and plotted as a function of the concentration of the respective catalytic entity to obtain the max. k_obs and K_M(STO) reported in Table 1.