Elucidation of structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein

Abstract

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5' leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae-RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein-protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.

Figure 1.

Similarities and differences in the 3-D models of the specificity (S) domain of Mja and yeast RNase P (10,41). L7Ae (whose paralog is POP3 in yeast), RPP21, and RPP29 (whose homolog is POP4 in yeast) form a heterotrimer when bound to the RPR’s S domain. The RPPs are depicted as surface representations while the RPR is depicted as cartoon; the catalytic (C) and specificity (S) domains are colored in silver and gray, respectively. (A) L7Ae in Mja RNase P is engaged in RNA–protein interactions as it binds a kink-turn in the P12 region of RPR and in protein–protein interactions by direct contacts with RPP21. (B) Since the P12 region in yeast RPR does not possess a kink-turn, POP3 (paralog of L7Ae) is assembled into the holoenzyme largely by taking advantage of protein–protein interactions with RPP21. Structural images were made with Pymol [4.6 Å model, PDB 6K0A (A) and 3.5 Å model, PDB 6AH3 (B)].

Figure 2.

Analysis of Methanocaldococcus jannaschii (Mja) RPR by size-exclusion chromatography. (A) Secondary structure of Mja RPR. P, consecutively numbered paired region; K-turn, kink-turn. The gray box in the P12 helix shows the two kink-turns, KT2 and KT3, that are predicted to be bound by Mja L7Ae (31). The gray box in the P1 stem shows the region that is absent in the RPR used in the cryo-EM study (41). The 5′- and 3′-terminal overhangs (colored) show nucleotides in the native Mja RPR that were identified by RACE experiments reported here. These additional nucleotides were not included in the RPRs used here or any previous study. (B) Three samples corresponding to the purified Mja RPR with (orange line, ) or without (blue line, ) the addition of Mja L7Ae during the in vitro transcription reaction, or the purified Mja RPR assembled with Mja L7Ae post-transcription (green line, ) were separately loaded onto the Superdex 200 increase 10/30 GL column (GE Healthcare) at a flow rate of 1 ml/min. Eluent was monitored for absorbance at 280 nm. Fractions corresponding to the main peak were collected and concentrated for native MS analysis.

Figure 3.

Full native mass spectra of the different Mja RNase P assemblies.Mass spectra are annotated with colored shapes that mark each charge state distribution and the charge state of the most abundant peak in each distribution. The oligomer assignments for each charge state distribution are displayed next to the corresponding-colored shape, with the same labeling scheme for all four spectra. Mass spectra of (A) MjaRPR+L7Ae (SEC fraction), (B) Mja RPR+L7Ae assembled with Mja RPP21·RPP29, (C) Mja RPR+L7Ae assembled POP5·RPP30 and (D) Mja RPR+L7Ae assembled with both RPP21·RPP29 and POP5·RPP30. In (C, D), isolation and fragmentation spectra of the 8500–11000 m/z region are also shown. The CID voltage is ∼150–180 V by HCD. Oligomer assignments are displayed below the spectra with the corresponding-colored shapes used to annotate each charge state distribution. The pink boxes represent the quadruple isolation ranges used for the fragmentation spectra.

Figure 4.

Mass photometry (MP) analysis of Mja RNase P. (A) Mja RPR (1 μM) was refolded and diluted in 800 mM NH₄OAc and 2 mM Mg(OAc)₂ before being loaded onto the mass photometer (Refeyn). (B–D) Mja RPR (1 μM) was refolded and reconstituted with a 4-fold molar excess of Mja L7Ae (4 μM), a two-fold molar excess of Mja RPP21·RPP29 (2 μM) and Mja POP5·RPP30 (2 μM) in 800 mM NH₄OAc and 2 mM Mg(OAc)₂. The samples were diluted before MP analysis. The expected masses for the holoenzyme monomer and dimer are based on the mass of [1 RPR + 1 RPP21 + 1 RPP29 + 1 POP5 + 1 RPP30 + 2 L7Ae] and [2 RPR + 2 RPP21 + 2 RPP29 + 2 POP5 + 2 RPP30 + 4 L7Ae] species, respectively. Although we obtained triplicate measurements, the data from only a single measurement are shown here (see Supplementary Table S2 for the data from the other runs). (E, F) Mja RPR (1 μM) was refolded and reconstituted with either a four-fold molar excess of Mja L7Ae (4 μM) and a two-fold molar excess of Mja POP5·RPP30 (2 μM) or only a 2-fold molar excess of Mja POP5·RPP30 (2 μM). The assemblies were in 800 mM NH₄OAc and 2 mM Mg(OAc)₂. The expected masses for the monomer and dimer are based on the mass of [1 RPR + 1 POP5 + 1 RPP30 ± 2 L7Ae] and [2 RPR + 2 POP5 + 2 RPP30 ± 4 L7Ae] species, respectively. In all cases, the observed masses for each population were calculated by the DiscoverMP software (Refeyn) and the % count for each population is shown in parentheses.

Figure 5.

Comparison of the activity of Mja RNase P reconstituted with either RPR (WT) or its kink-turn mutant derivatives. Ten nM RPR (WT, mKT2, mKT3, mKT23) was assembled with 100 nM RPP21·RPP29, 100 nM POP5·RPP30, and varying amounts of L7Ae (0, 100, 250 or 500 nM). Cleavage assays were performed in 50 mM HEPES–KOH (pH 8), 800 mM (NH₄)OAc, 2 mM Mg(OAc)₂, using as substrate 500 nM E. coli pre-tRNA^Tyr spiked with a trace amount of 5′-[³²P]-labeled E. coli pre-tRNA^Tyr. Data from three independent measurements were used to determine the mean ± standard deviation for the reported turnover numbers.

Figure 6.

Native mass spectra of partial RNase P assemblies consisting of RPR_mKT23 with L7Ae, RPP21·RPP29, or RPP21·RPP29 + L7Ae. Mass spectra are annotated with colored shapes that mark each charge state distribution and the charge state of the most abundant peak in each distribution is labeled. The oligomer assignments for each charge state distribution are displayed next to the corresponding-colored shape, with the same labeling scheme for all four spectra. All reconstitutions were for 10 min at 55°C before native MS analysis. Mass spectra of (A) 1 μM RPR_mKT23, (B) 1 μM RPR_mKT23 + 1 μM L7Ae, (C) 1 μM RPR_mKT23 + 2 μM RPP21·RPP29 and (D) 1 μM RPR_mKT23 + 2 μM RPP21·RPP29 + 1 μM L7Ae (assembled in this order). In (D), we do observe a small amount of the [RPR_mKT23 + RPP21·RPP29 + L7Ae]₂, whose significance is unclear.