Eukaryotic ribonucleases P/MRP: the crystal structure of the P3 domain

Abstract

Ribonuclease (RNase) P is a site-specific endoribonuclease found in all kingdoms of life. Typical RNase P consists of a catalytic RNA component and a protein moiety. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme, RNase MRP, which has similar components but has evolved to have different specificities. The eukaryotic RNases P/MRP have acquired an essential helix-loop-helix protein-binding RNA domain P3 that has an important function in eukaryotic enzymes and distinguishes them from bacterial and archaeal RNases P. Here, we present a crystal structure of the P3 RNA domain from Saccharomyces cerevisiae RNase MRP in a complex with RNase P/MRP proteins Pop6 and Pop7 solved to 2.7 A. The structure suggests similar structural organization of the P3 RNA domains in RNases P/MRP and possible functions of the P3 domains and proteins bound to them in the stabilization of the holoenzymes' structures as well as in interactions with substrates. It provides the first insight into the structural organization of the eukaryotic enzymes of the RNase P/MRP family.

Figure 1

The RNA components of eukaryotic RNase P and RNase MRP have multiple common structural features including the P3 domain. (A) Secondary structure diagram of the RNA component of S. cerevisiae RNase P. (B) Secondary structure diagram of the RNA component of S. cerevisiae RNase MRP. (C) The modified P3 domain of RNase MRP used in crystallization (the mutated and artificially introduced nucleotides are italicized). The diagrams and the nomenclature of the structural elements are based on Frank et al (2000); Li et al (2002); Walker and Avis (2004) and Esakova et al (2008).

Figure 2

(A, B) Stereoscopic views of the P3 RNase MRP RNA domain in complex with the RNase MRP/RNase P protein components Pop6 and Pop7. The RNA nucleotides 29–50 are shown in red; nucleotides 59–78 are shown in green; the protein component Pop6 is shown in blue; the protein component Pop7 is shown in yellow; H0, H1, H2-α-helices; E0, E1, E2, E3, E4-β-strands; the positions of the N- and C-terminal ends of the proteins are marked correspondingly. The disordered loops in Pop6 (residues 122–128) and Pop7 (residues 105–124) are symbolized by blue and yellow dots, respectively; the artificially introduced GAAA tetraloop linker connecting the nucleotides 29 and 78 in the crystallization construct is not shown.

Figure 3

The folds of the RNase P/RNase MRP components Pop6 and Pop7 resemble those found in proteins of the Alba family. H0, H1, H2-α-helices; E0, E1, E2, E3, E4-β-sheets. (A) Pop6. (B) Pop7. (C) Sulfolobus solfataricus Alba protein Sso10b (PDB code 1h0x), a typical Alba protein (Wardleworth et al, 2002). (D) A structure-guided sequence alignment for Pop6 (lines 2–10) and Pop7 (lines 11–19) versus Alba protein Sso10b (lines 20–21). The alignments are shown for S. cerevisiae (Sc, lines 5 and 14), Zygosaccharomyces rouxii (Zr, lines 6 and 15), Candida glabrata (Cg, lines 7 and 16), Kluyveromyces lactis (Kl, lines 8 and 17), Xenopus laevis (Xl, lines 9 and 18) and human (Hs, lines 10 and 19) proteins. Line 1: secondary structure elements; lines 2, 11 and 20: secondary structures of Pop6, Pop7 and Alba Sso10b (PDB 1h0x), respectively. The residues conserved in Pop6 (lines 5–10), Pop7 (lines 14–19) and Alba proteins (line 21) are highlighted as follows: non-polar aliphatic (GAPVLIM, yellow), aromatic (FYW, dark blue), polar uncharged (STCNQ, green), positively charged (KHR, light blue), negatively charged (DE, brown). The absolutely conserved glycine is highlighted in red. The shown conservation pattern for Alba is based on the alignment of Alba proteins with known structures (PDB ID 1h0x, 1y9x, 1nfj, 1nh9, 2h9u, 2z7c, 2bky, the alignment is not shown). Residues involved in interactions with the P3 domain RNA are marked with asterisks in lines 3 and 12; residues involved in the formation of the Pop6/Pop7 heterodimer are marked with asterisks in lines 4 and 13. For the complete sequence alignment see Supplementary Figure S2.

Figure 4

Interactions in the P3 domain. (A) RNA–protein interactions. Pop6 residues are highlighted in blue; Pop7 residues are highlighted in orange. P3 domain nucleobases are shown as boxes; P3 loop nucleobases exposed to the solvent are highlighted in green. Van der Waals interactions and hydrogen bonding are shown by arrows; the lines parallel to the nucleobase boxes show stacking interactions. (B) Sequence-specific interaction between A39 and Gln89 in Pop6. (C) A37 is involved in extensive interactions with Pop7. (D) Absolutely conserved Gly69 in Pop7 allows for a sharp turn. (E) A stack of hydrophobic residues (Ile33 (β-strand E1), Val65 (β-strand E2), Val92 (β-strand E3) and Leu136 (β-strand E4)) helps stabilize the β-sheet in Pop7.

Figure 5

The electrostatic potential of the surface of the Pop6/Pop7 protein heterodimer. The positive charges are located predominantly in the P3 RNA-binding region. Positively charged areas are shown in blue, neutral are shown in white and negatively charged are shown in red. The P3 RNA domain nucleotides 29–50 are shown in red; nucleotides 59–78 are shown in green.

Figure 6

Modelling S. cerevisiae RNase MRP P3 RNA domain into the crystal structure of B. stearothermophilus RNase P (PDB ID 2A64). The P3 RNA domain and proteins bound to it are positioned in the vicinity of RNA structural elements that serve to stabilize the tertiary fold in bacterial RNase P (Torres-Larios et al, 2006), but are missing in eukaryotic enzymes. (A) A surface representation of B. stearothermophilus RNase P; helical stem P3 is shown in green; stems P15.1 and P15.2 (which stabilize the tertiary structure of bacterial RNase P, but are missing in eukaryotic enzymes) are shown in red. (B) A chimerical model built of B. stearothermophilus RNase P with stems P15.1, P15.2 removed, and S. cerevisiae RNase MRP P3 domain replacing the bacterial P3 stem. Bacterial RNase P is shown in grey; S. cerevisiae P3 domain RNA (inserted by superposition of the proximal helical stem of the eukaryotic P3 RNA domain and the bacterial helical stem P3) is shown in green; Pop6 is shown in blue and Pop7 is shown in yellow.