Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

Abstract

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.

Figure 1.

(A) A modified Pyrococcus furiosus RNase P RNA (Pfu RPR ext mP1) labeled with both Cy3 and Cy5 fluorophores. To generate Pfu RPR ext mP1, both the 5′- and 3′-termini were extended (inset, right) while the two terminal bp in the P1 helix were deleted. The cytidine at position 170 (wild-type numbering) was replaced with an amine-modified uridine (red) to facilitate internal Cy5 labeling via splint ligation (see Figure 2), and a Cy3-labeled DNA oligonucleotide was annealed to the 5′-end to obtain a dual-labeled RPR. (B) General scheme for Förster resonance energy transfer (FRET) studies. Increasing [Mg²⁺] is expected to promote interactions between the C (dark gray) and S (light gray) domains, thereby increasing the proximity of the Cy3 (donor) and Cy5 (acceptor) fluorophores and FRET efficiency.

Figure 2.

Use of splint ligation to internally label Pfu RPR ext mP1 with a Cy5 fluorophore. (A) The RPR was synthesized in three fragments: 5′-fragment (5-F, transcribed in vitro), internal fragment (I-F, commercially synthesized), and 3′-fragment (3-F, transcribed in vitro). Each fragment was processed individually prior to a two-step ligation using T4 RNA ligase 2 and a DNA splint. Mass spectrometry was used to verify the masses of the (B) 5′ fragment [5-F], post-RNase H cleavage; (C) 3′ fragment [3-F], post-RppH treatment; and (D) final Pfu RPR ext mP1 labeled with Cy5. Charge state distributions for the most intense species in each sample are indicated with diamonds, and the main charge state is labeled. In each spectrum, the species of intended length (N) is shown in blue while any prominent, 3′-extended species (N+#) are marked in varying shades of gray. Representative regions of low-intensity peaks that are indicative of additional heterogeneity have been denoted with an asterisk. In all spectra, the y-axis (not shown) represents relative intensity.

Figure 3.

Representative microscale thermophoresis (MST) measurements of Cy5-labeled oligonucleotide (Cy5–oligo) binding to (A) unlabeled Pfu RPR ext mP1 at 10 mM (⚬, solid line) and 500 mM (•, dashed line) Mg²⁺, and (B) Pfu RPR ext mP1 + 5 RPPs at 0.033 mM (□, solid line) and 30 mM (▪, dashed line) Mg²⁺. Plots were fit to a hyperbolic binding isotherm to yield K_D values. In the inset tables, mean and standard deviation values were calculated from three technical replicates (see Supplementary Figure S3 for the primary data from individual trials).

Figure 4.

Effect of Mg²⁺ on the activity and inter-domain interactions of Pfu RPR ext mP1. (A) Representative data showing cleavage of γ-³²P-ATP-labeled Thermus thermophilus (Tth) pre-tRNA^Gly by unlabeled Pfu RPR ext mP1 in 450 mM Mg²⁺ and at 37°C during a 20-min time-course (also, see Supplementary Figure S4 and S5). (B) Mg²⁺-dependent increase in the activity of unlabeled Pfu RPR ext mP1. Average turnover numbers calculated from four independent initial velocity measurements are plotted against [Mg²⁺]. (C) Mg²⁺-dependent increase in FRET efficiency for dual-fluor-labeled Pfu RPR ext mP1. Average FRET efficiencies obtained from three independent measurements are plotted against [Mg²⁺] (see Supplementary Figure S6 for additional data). (D) Overlay of turnover number (solid line) and FRET efficiency (dashed line), which shows that FRET efficiency correlates with the rate of catalysis as a function of [Mg²⁺]. Relative activity and E_FRETvalues were obtained by normalization against the highest turnover number or FRET efficiency, respectively; since this reference value was not always at the same concentration of Mg²⁺ among the replicates, there is minor variability between panels B or C versus D but the trends are highly similar. In instances where error bars are not seen, the errors are smaller than the symbols used.

Figure 5.

Effect of Mg²⁺ on the activity and inter-domain interactions of Pfu RPR ext mP1 reconstituted with all 5 RPPs. (A) Representative data showing cleavage of γ-³²P-ATP-labeled Escherichia coli (Eco) pre-tRNA^Tyr by unlabeled Pfu RPR ext mP1 + 5 RPPs in 30 mM Mg²⁺ and at 37°C during a 5-min time-course. For illustrative purposes, the gel was cropped to reposition non-adjacent lanes of interest; no other modifications were made to the image. (B) Mg²⁺-dependent increase in the activity of unlabeled Pfu RPR ext mP1. Average turnover numbers calculated from three independent initial velocity measurements are plotted against [Mg²⁺], and the data were fit to the Hill equation. Dual-fluor-labeled Pfu RPR ext mP1 + 5 RPPs (C) without and (D) with Eco pre-tRNA^Tyr shows a decrease in FRET from low (0.5 mM; purple) to high (30 mM; red) Mg²⁺. A representative spectrum from one trial at each Mg²⁺ concentration is shown. Within each set, emission from direct Cy5 excitation was normalized against the sample with the highest signal. The same scaling factor was then applied to the corresponding emission spectrum obtained from Cy3 excitation (see Supplementary Figure S10 for additional data). (E) Mg²⁺-dependent increase in FRET efficiency for dual-fluor-labeled Pfu RPR ext mP1 + 5 RPPs without (dotted line) and with (solid line) Eco pre-tRNA^Tyr. Average FRET efficiency from three independent measurements is plotted against [Mg²⁺]. (F) Comparison of relative turnover numbers (black) and ΔFRET values (green solid line). Data for Pfu RPR ext mP1 + 5 RPPs + Eco pre-tRNA^Tyr were fit to the Hill equation to obtain the n_H (Hill coefficient) and K_1/2Mg²⁺ values in the inset (see Supplementary Figure S11 for additional data).

Figure 6.

Model of potential structural changes in archaeal RNase P (also, see Scheme I). Based on the ensemble FRET measurements, the RPR (gray) alone likely adopts an ‘open’ conformation (low FRET efficiency; A, left) at 10 mM Mg²⁺ while the complex of Pfu RPR + 5 RPPs exists in a ‘constrained’ conformation (high FRET efficiency; B, left) at 0.033 mM Mg²⁺. Neither state is competent for pre-tRNA cleavage (C, left). With increasing [Mg²⁺], both the RPR and the holoenzyme complex comprised of Pfu RPR + 5 RPPs undergo structural remodeling to converge on a common ‘closed’ active conformation (A–C, right). While there are caveats to using ensemble FRET measurements to establish a precise distance between two fluorophores, conservation of the active site and pre-tRNA anchors in different RNase P RNP variants provide some support for this qualitative model generated using the cryo-EM structure of Methanocaldococcus jannaschii (Mja) RNase P as template (40). Although we depict single conformations in this model for the sake of simplicity, each state is more accurately described by an ensemble. E_FRET classified using 0.03 < low < 0.10 < medium < 0.30 < high < 0.60.