Crystal structure of an RNA/DNA strand exchange junction

Figures

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  Fig 1. Stabilizing features of the crystal lattice. (A) Crystallization construct sequence. Black, DNA; red, RNA. (B) Schematized drawing (not to scale) of the crystal lattice along a direction that depicts the helical network formed by Molecules 1 and 2. Green shading, Molecule 1; blue shading, Molecule 2; orange shading, Molecule 3 (cross section). (C) Similar to panel B, but along a direction that depicts the helical network formed by Molecule 3. (D) Asymmetric unit colored by atomic B-factor. The thickness of the cartoon model also reflects the local B-factors. (E) Model and 2mFo-DFc map (sharpened by -38 Å2, displayed at 3.3σ) of the Ade-Ade and Cyt-Cyt base pairs (contributed by the flap nucleotides of Molecules 1 and 2) that bridge the helical network formed by Molecule 3. Distortion in the map is due to diffraction anisotropy (see Methods).

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  Fig 2. Molecule-to-molecule similarity and hydrogen bonding at the flapped nucleotides. (A) All-atom alignment of the three molecules in the asymmetric unit. Green, Molecule 1; blue, Molecule 2; orange, Molecule 3. Molecules 2 and 3 were aligned to Molecule 1 in this depiction. (B) Hydrogen bonding at the flapped nucleotides of Molecule 1. Dotted lines indicate hydrogen bonds, and adjacent numbers indicate interatomic distance in Å. Black, DNA; red, RNA. This hydrogen bonding pattern is also observed in Molecule 2 but not in Molecule 3.

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  Fig 3. Alignments to regularized A-form/B-form helices. (A) Black, DNA of Molecule 1; red, RNA of Molecule 1; white, regularized B-form DNA:DNA helix aligned to the 6 bp of Molecule 1’s DNA:DNA helix; pink, regularized A-form DNA:DNA helix aligned to the 6 bp of Molecule 1’s RNA:DNA helix. (B) Cartoon depiction, focused on the continuous strand. The alignment procedure for each 6-bp block was identical to that performed in panel A, but in this depiction, the B-form (white) and A-form (pink) helices were extended by an additional 6 bp (extended nucleotides were not considered during alignment) to illustrate the path that the helix would take if continuing along a perfect B-form or A-form trajectory. EP, exchange point (that is, the phosphodiester or gap lying between the two nucleotides where the helix changes from RNA:DNA to DNA:DNA). (C) Similar to panel B, but focused on the discontinuous strand. (D) Close-up of the same representation depicted in panel A, focused on the nucleotides that deviate most dramatically from the aligned A-form helix.

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  Fig 4. Geometric details of the A-to-B transition. (A) For a given base step, the parameter zP is the mean of the z-displacement of the two phosphorus atoms from the dimer’s reference xy-plane. Note that zP is defined by a pair of dinucleotides, so there are only 11 data points for a 12-bp helix, and integral x-values lie between the base pairs in the diagram. This parameter was originally introduced for its utility in distinguishing A-form from B-form base steps. Black, DNA; red, RNA. (B) χ and δ are the two nucleotide torsion angles that best distinguish A-form from B-form geometry. Note that these torsion angles are defined for each individual nucleotide, so there are 24 data points for a 12-bp helix. Integers in red refer to individual nucleotides, as indicated in the schematic at the bottom. Dashed ellipses were drawn to match those depicted in [27]. (C) Y-displacement. Similar to zP, this parameter describes base steps (pairs of dinucleotides), not individual nucleotides. This parameter cannot distinguish A-form from B-form geometry. Instead, note that the base step across the exchange point dramatically departs from both A-form and B-form geometry.

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  Fig 5. Structural principles of A-on-B stacking at the RNA-5′/DNA-3′ strand exchange junction. (A) Simplified schematics illustrating strand-specific compression and tilting of the helical axes. The slanted appearance of the RNA:DNA duplex is intended to represent the base pair inclination characteristic of A-form duplexes, which pushes the 3′ DNA end farther along the helical axis than the 5′ RNA end. Black, DNA; red, RNA. (B) Helical cross-sections. Black, DNA:DNA helix; red, RNA:DNA helix. The rectangle represents the base pair nearest the exchange point (centered in the B-form helix, off-center in the A-form helix). The solid circle represents the helical axis. The true stacking solution is a combination of the three principles illustrated here, although Molecule 3 does not exhibit tilting.