Dissecting Monomer-Dimer Equilibrium of an RNase P Protein Provides Insight Into the Synergistic Flexibility of 5’ Leader Pre-tRNA Recognition

Ribonuclease P (RNase P) is a universal RNA-protein endonuclease that catalyzes 5’ precursor-tRNA (ptRNA) processing. The RNase P RNA plays the catalytic role in ptRNA processing; however, the RNase P protein is required for catalysis in vivo and interacts with the 5’ leader sequence. A single P RNA and a P protein form the functional RNase P holoenzyme yet dimeric forms of bacterial RNase P can interact with non-tRNA substrates and influence bacterial cell growth. Oligomeric forms of the P protein can also occur in vitro and occlude the 5’ leader ptRNA binding interface, presenting a challenge in accurately defining the substrate recognition properties. To overcome this, concentration and temperature dependent NMR studies were performed on a thermostable RNase P protein from Thermatoga maritima. NMR relaxation (R1, R2), heteronuclear NOE, and diffusion ordered spectroscopy (DOSY) experiments were analyzed, identifying a monomeric species through the determination of the diffusion coefficients (D) and rotational correlation times (τc). Experimental diffusion coefficients and τc values for the predominant monomer (2.17 ± 0.36 * 10−10 m2/s, τ c = 5.3 ns) or dimer (1.87 ± 0.40* 10−10 m2/s, τ c = 9.7 ns) protein assemblies at 45°C correlate well with calculated diffusion coefficients derived from the crystallographic P protein structure (PDB 1NZ0). The identification of a monomeric P protein conformer from relaxation data and chemical shift information enabled us to gain novel insight into the structure of the P protein, highlighting a lack of structural convergence of the N-terminus (residues 1–14) in solution. We propose that the N-terminus of the bacterial P protein is partially disordered and adopts a stable conformation in the presence of RNA. In addition, we have determined the location of the 5’ leader RNA in solution and measured the affinity of the 5’ leader RNA–P protein interaction. We show that the monomer P protein interacts with RNA at the 5’ leader binding cleft that was previously identified using X-ray crystallography. Data support a model where N-terminal protein flexibility is stabilized by holoenzyme formation and helps to accommodate the 5’ leader region of ptRNA. Taken together, local structural changes of the P protein and the 5’ leader RNA provide a means to obtain optimal substrate alignment and activation of the RNase P holoenzyme.

and low (153 μM) concentrations at 318 K. Spectra are overlapped by chemical shifts, with high (red) and low (blue) concentrations identified. Contour levels are rendered such that peaks of non-dominant species are observable. Slow exchange kinetics between the monomer-dimer equilibrium reveals distinct chemical shifts and potential conformational difference of P protein at high and low concentrations. A total of 48 residues were confirmed to contain distinct chemical shifts at high and low protein concentrations (listed in Supplement Table 1).
Supplemental Figure 2. Temperature-dependent 1 H, 15 N-HSQC spectra of T. maritima P protein at 153 μM. Spectra were acquired at 35 o C, 45 o C, 55 o C and 65 o C (light to deep blue gradient, respectively). At 153 µM, the annotated resonances represent the dominant monomeric conformer, while the dimer conformer is represented by the secondary, very weak peak intensities. As the temperature increases, all peaks undergo uniform chemical shift changes and no obvious peak intensity changes occur. This suggests the monomer-dimer equilibrium in solution is largely independent of temperature.
Supplemental Figure 3. Sequential NOE connectivities of the T. maritima RNase P protein reveal the extent of secondary structure in solution. Strip plots from the analyzed 3D 15 N-NOESY HSQC spectrum include amino acid regions R59-R68 (3 helix) (A), residues R8-R12 (random coil) (B), and residues L18-K22 (1 helix) (C). The x-axis is the proton dimension of the amide (HN) resonance and the y-axis is the proton dimension that includes H − N NOEs. The strip plots were assigned via oneletter amino acid codes. The 3D 15 N-NOESY HSQC spectrum was collected at 318 K with a 120 ms mixing time. In the dimer, R14 interacts with D16 from a different molecule, forming a salt bridge. In addition, E23 and R52 form an electrostatic interactions along the α1 helix. These specific dimer contacts in T. maritima P protein likely help to strengthen a hydrophobic stacking interaction of conserved F17 residues. These residues occlude 5' leader RNA binding when a stable dimer is formed. Left and right panels show orthogonal views of the interface from P protein molecules (olive and dark olive cartoons). Residues that contribute to dimerization are highlighted: charged residues providing electrostatic stabilization are blue (positive) or red (negative), while hydrophobic residues forming aromatic stacking interactions are green. Side chain contacts (sticks) emphasize the intermolecular dimer contacts.

Supplemental Figure 5:
Mapping residues that contatin different chemical shifts due to monomer-dimer P protein equilibrium. The residues listed in supplement table 1 contain distinct chemical shifts for the monomer and dimer conformers are highlighted in purple on the crystal structure of P protein (PDB:1NZ0).
Supplemental Figure 6. Single-stranded RNA binding to the T. maritima RNase P protein. The 15 N HSQC of the 153 M T. maritima RNase P protein is shown (central) and clockwise (1-5) are zoomed-in regions of the spectra to gauge residues that shift or are unchanged due to RNA binding. During titrations, the leader RNA was gradually added with increased molar ratios of 0:1 (red), 0.2:1 (orange), 0.4:1 (purple), 1:1 (blue), and 2:1 (green) to the 153 μM protein sample. Each region contains labeled single-letter amino acid residues that are influenced by the titration experiment. Figure 7. Single-stranded RNA binding to the 153 µM T. maritima RNase P protein reveals some dimer shifted peaks and unchanged monomer peaks. Examples of dimer conformer peaks (Q28 and F82) that shift upon RNA addition, as well as monomer conformer peaks (F34 and G55) that do not shift upon RNA addition. The leader RNA was gradually added at increased molar ratios of 0:1 (red), 0.4:1 (purple), and 2:1 (green) to the 153 μM protein sample. For the Q28 dimer peak, it is possible that RNA addition may shift equilibrium towards the Q28 monomer. For the F82 dimer peak, RNA addition shifts the dimer peak away from a monomer F82 peak and likely does not shift equilibrium towards the monomer conformation. Additional experiments would be required to determine whether specific RNA interactions with the dimer P protein shifts the equilibrium towards the monomeric conformation.