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 (R-1, R-2), 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 (tau(c)). Experimental diffusion coefficients and tau(c) values for the predominant monomer (2.17 +/- 0.36 * 10(-10) m(2)/s, tau(c) = 5.3 ns) or dimer (1.87 +/- 0.40* 10(-10) m(2)/s, tau(c) = 9.7 ns) protein assemblies at 45 degrees 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.

Automated summary

RNase P is a crucial RNA-protein endonuclease for processing ptRNA. The functional RNase P holoenzyme consists of a single P RNA and a P protein. Studies have shown that at 45 degrees Celsius, the diffusion coefficients and rotational correlation times of RNase P protein monomers and dimers correlate well with calculated diffusion coefficients from crystallographic data.