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. 1999 Mar;6(3):285-92.
doi: 10.1038/6722.

Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot

L Su  1 , L Chen, M Egli, J M Berger, A Rich
Collaborators, Affiliations

Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot

L Su et al. Nat Struct Biol. 1999 Mar.

Abstract

Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. A pseudoknot has two stems that form a quasi-continuous helix and two connecting loops. A 1.6 A crystal structure of the beet western yellow virus (BWYV) pseudoknot reveals rotation and a bend at the junction of the two stems. A loop base is inserted in the major groove of one stem with quadruple-base interactions. The second loop forms a new minor-groove triplex motif with the other stem, involving 2'-OH and triple-base interactions, as well as sodium ion coordination. Overall, the number of hydrogen bonds stabilizing the tertiary interactions exceeds the number involved in Watson-Crick base pairs. This structure will aid mechanistic analyses of ribosomal frameshifting.

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Figures

Figure 1
Figure 1. Beet western yellow virus (BWYV) pseudoknot electron density maps at 1.9 Å resolution contoured at 1.0σ above the mean.
a, View of the refined structure in the minor-groove RNA triplex region superimposed on the solvent-flattened experimental electron density map. Loop 2 is shown in green, stem regions in gold, loop 1 in red. b, Detailed view of the refined structure superimposed on the same map in the major groove at the stem 1–loop 1 junction. Stems are in gold, C8 in red, A25 in magenta. In both maps 2'-OH and phosphate oxygen density, as well as ring characteristics of base density are clearly visible. These figures were generated with the program O (ref. 46 ).
Figure 2
Figure 2. The beet western yellow virus (BWYV) pseudoknot crystal structure and secondary sequence.
a, Stereo view of the pseudoknot crystal structure. The color scheme of bases corresponds to that used in the secondary-structure diagram in (b). Loop 1 (red) crosses the major groove of stem 2, and loop 2 (green) stacks in the minor groove of stem 1. The stem backbone is blue; bases are gold. b, The conventional representation of the BWYV pseudoknot secondary sequence. Stem 1 of the wild-type pseudoknot sequence starts at C3, G1 was added at the 5' end to assist transcription and G2 is in the original mRNA sequence. The crystal structure has significant differences from the secondary prediction: A25–U13 is not paired, and the A25–G28 strand in stem 2 is flipped to the other side. c, Stereo view at a slightly different angle from (a) of stem 1 and stem 2 without the loop nucleotides. Stem 2 is rotated in relation to stem 1 and is noncolinearly stacked; all base pairs are highly propeller twisted. At the junction, G12 is stacked on C14, but on the opposite strand the bottom base A25 of stem 2 (magenta) is not stacked on G7, the top base of stem 1. d, The general fold of the pseudoknot and metal ions. A magnesium ion in rose color binds at the 5'-triphosphate region. One sodium ion in orange is coordinated in the minor groove.
Figure 3
Figure 3. RNA triplex interactions of loop 2 in the minor groove of stem 1.
a, Separate view of the conserved predominantly adenosine ladder. Despite the systematic stacking, each loop base is rotated in different orientations to maximize interactions with the groove nucleotides. b, The tilted A20 interacts with two layers of base pairs through a base triplet, and a 2'-OH multiple hydrogen-bonding network. For clarity, the ribose of C5 is omitted, but the weaker hydrogen bond between N7 of A20 and C5 2'-OH is indicated. c, A21 and C22 contact G16 through another 2'-OH multiple interaction. The hydrogen bond from the C22 amino group is relatively weaker. d, A23 forms a triple-base interaction where the 2'-OH of C15 interacts with N1 of A23. e, A24 forms a unique interaction with G7. Not shown is a hydrogen bond between the 2'-OH of A24 and the O4' of the A25 furanose ring.
Figure 5
Figure 5. Stereo view of stabilizing interactions at sharp turns, ions and water molecules.
a, The 'C turn' from loop 1 (red) to C10 of stem 2 (gold) is stabilized by a base-to-phosphate hydrogen bond (magenta dashed lines) and an organized water network (cyan spheres). b, An array of water molecules in the major groove of stem 2 (gold) stabilize loop 1 (red) on the other side. The water molecules stabilize the insertion of C8 into the major groove to form quadruple-base interactions. Junctional base A25 is in magenta, and A24 of loop 2 is in green. c, A sodium ion in the minor groove mediates base-to-base contacts between stem 1 (gold) and loop 2 (green). The sodium ion (orange sphere) coordinates to N3 and 2'-OH of G16, pro-Rp phosphate oxygen and more weakly to N7 of A21. A water molecule that hydrogen-bonds to N4 of C22 is also coordinated. Two other water molecules mediate 2'-OH, phosphate and base contact. Dashed lines are hydrogen bonds, and solid lines are metal coordination. All diagrams except Fig. 1 were generated with the program RIBBONS.
Figure 4
Figure 4. Quadruple-base interactions of loop 1.
a, The C8 organizer base of loop 1 inserts deeply into the major groove of stem 2 and interacts simultaneously with bases of three other nucleotides. The asterisk next to N3 of C8 indicates probable protonation with a hydrogen bond to O6 of G12. C26 is propeller twisted in the C26–G12 base pair as a result of stacking on A25. b, A25 does not pair with its predicted basemate U13, but is involved in the quadruple-base interaction, and tilts between the C8 and C14 layer.
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