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. 2011 Oct;17(10):1870-83.
doi: 10.1261/rna.2880311. Epub 2011 Aug 30.

The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate

Blaine H M Mooers  1 , Amritanshu Singh
Affiliations

The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate

Blaine H M Mooers et al. RNA. 2011 Oct.

Abstract

Guide RNAs bind antiparallel to their target pre-mRNAs to form editing substrates in reaction cycles that insert or delete uridylates (Us) in most mitochondrial transcripts of trypanosomes. The 5' end of each guide RNA has an anchor sequence that binds to the pre-mRNA by base-pair complementarity. The template sequence in the middle of the guide RNA directs the editing reactions. The 3' ends of most guide RNAs have ∼15 contiguous Us that bind to the purine-rich unedited pre-mRNA upstream of the editing site. The resulting U-helix is rich in G·U wobble base pairs. To gain insights into the structure of the U-helix, we crystallized 8 bp of the U-helix in one editing substrate for the A6 mRNA of Trypanosoma brucei. The fragment provides three samples of the 5'-AGA-3'/5'-UUU-3' base-pair triple. The fusion of two identical U-helices head-to-head promoted crystallization. We obtained X-ray diffraction data with a resolution limit of 1.37 Å. The U-helix had low and high twist angles before and after each G·U wobble base pair; this variation was partly due to shearing of the wobble base pairs as revealed in comparisons with a crystal structure of a 16-nt RNA with all Watson-Crick base pairs. Both crystal structures had wider major grooves at the junction between the poly(U) and polypurine tracts. This junction mimics the junction between the template helix and the U-helix in RNA-editing substrates and may be a site of major groove invasion by RNA editing proteins.

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Figures

FIGURE 1.
FIGURE 1.
(A) Base-pairing between the A6 mRNA (top strand) and the gA6[14] guide RNA (bottom strand). The asterisks mark the uridylates that are deleted by editing. The anchor helix is to the right of the editing site, and the U-helix is to the left. The template sequence in the middle of the guide RNA is represented by a loop. The box outlines the fragment of the U-helix that was crystallized. (B) The fragment was fused head-to-head with a duplicate fragment to give the RNA hexadecamer on the right. (C) The G·U base pairs in the U-helix were replaced with G–C base pairs to give the WC-helix on the left. Two copies of the WC-helix were fused head-to-head to give the 16-nt RNA on the right. (D) Triple base-pair motifs possible in U-helices. The first motif (i) occurred in the crystal structure of the U-helix.
FIGURE 2.
FIGURE 2.
Comparisons of the U-helix and WC-helix RNA structures. Electron density (2m|Fo| – D|Fc|, 1.5 σ contour level) around the single-strand in the asymmetric unit of the crystal structures of the U-helix (A) and the WC-helix (B). (C) The crystal structure of the U-helix in black superposed on the crystal structure of the WC-helix in gray (RMSD = 0.78 Å when the G·U base pairs are excluded). The dark curve denotes the helical axis of the U-helix. The light curve denotes the helical axis of the WC-helix. (D) Probability distributions of the distances between corresponding atoms in the two superposed structures when all nucleotides (666 atoms) are used in the superposition (solid line) and when the six G·U base pairs are excluded (414 atoms) from the superposition (dashed line). (E) The direction cosines of the base-pair plane normal vector when the helical axis is aligned along the z-axis of the coordinate system.
FIGURE 3.
FIGURE 3.
Crystal packing of the U-helix RNA. (A) View down the c-axis of the R32 unit cell in the hexagonal setting (H32). The molecular dyad (data not shown) of the RNA duplex in the center of the unit cell lies on the twofold rotation axis that runs along the short diagonal of the a × b face of the unit cell. (B) Stereo view along the short diagonal in the a × b plane. (C) Stick model of two adjacent terminal base pairs in a stack of helices.
FIGURE 4.
FIGURE 4.
Base-pair parameters in the crystal structures of the U-helix (•) and the WC-helix (○): (A) Buckle, (B) propeller twist, (C) shear, (D) the relation between the change in base-pair shear and twist angle between adjacent base pairs. The line was fit to the U-helix data (R2 = 0.97). The base sequence (5′ to 3′) is along the x-axis for the U-helix (top) and WC-helix (bottom). Buckle and shear are inverted in sign about the molecular dyad in the center of the double helix (A,C).
FIGURE 5.
FIGURE 5.
Base-step parameters for the first eight base steps (values for the last seven base steps are identical to the first seven due to symmetry): (A) shift, (B) slide, (C) roll, and (D) twist.
FIGURE 6.
FIGURE 6.
Base stacking in the U-helix and WC-helix. Stereo views made with PyMOL of the third to eighth base steps: (A) A3pG4, (B) G4pA5, (C) A5pA6, (D) A6pU7, (E) U7pA8, and (F) A8pU9. Corresponding base steps from each structure are superposed using the C1′ and N1 or N9 atoms of the base pairs closest to the viewer. Residues in the opposing strand have asterisks. The residues numbers for the base pairs far from the viewer are in italics. The single letter residue code is not given when either a C or U occupy a site. The bonds of the U-helix structure are colored black. The bonds of the WC-helix structure are colored light gray. The right-handed screws of the helical axes project out of the plane of the page.
FIGURE 7.
FIGURE 7.
Electrostatic potential mapped onto solvent accessible surface (probe radius 1.4 Å) of the U-helix (A,C) and the WC-helix (B,D). The electrostatic potential ranges from −5 kbT/e to 1 kbT/e− because the negative potential of the major groove is dominant. The N2 nitrogen atoms of the guanines are represented by blue spheres scaled by 0.7 of their van der Waals radius. (A,B) Views toward the shallow groove (minor groove) in the center. (C,D) Views toward the deep groove (major groove) in the center. (E) Major groove widths—measured with 3DNA—of the U-Helix and the WC-helix by base step.
FIGURE 8.
FIGURE 8.
Metal binding sites in the crystal structures of the WC-helix (A,B) and U-helix (C,D). Distances are in Ångstroms. The figure in B is on a larger scale. The carbon atoms of symmetry-related molecules are colored green, black, and gray. The magnesium cation is colored green, and sodium and potassium cations are colored purple. (A) A magnesium-water complex [Mg2+(H2O)3] on a threefold crystallographic axis. The magnesium bound oxygen atoms of the phosphate of G7 and water 117. The distances between adjacent phosphates are shown. The typical distance in A-form RNA is 5.7 Å. (B) A sodium cation binds in the major groove with inner sphere coordination to the N7 nitrogen atom of A3. (C) A potassium cation sits on a crystallographic threefold axis and bridges three symmetry-related U13 phosphate O1P oxygen atoms. (D) A sodium–water complex [Na+(H2O)4] bound to the ribose O2′ hydroxyl of A5 and O4′ ring oxygen of A6 in the minor groove near a crystallographic threefold axis; the symmetry-related distances and labels are not shown.
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