A thymine tetrad in d(TGGGGT) quadruplexes stabilized with Tl+/Na+ ions

Abstract

We report two new structures of the quadruplex d(TGGGGT)4 obtained by single crystal X-ray diffraction. In one of them a thymine tetrad is found. Thus the yeast telomere sequences d(TG1-3) might be able to form continuous quadruplex structures, involving both guanine and thymine tetrads. Our study also shows substantial differences in the arrangement of thymines when compared with previous studies. We find five different types of organization: (i) groove binding with hydrogen bonds to guanines from a neighbour quadruplex; (ii) partially ordered groove binding, without any hydrogen bond; (iii) stacked thymine triads, formed at the 3'ends of the quadruplexes; (iv) a thymine tetrad between two guanine tetrads. Thymines are stabilized in pairs by single hydrogen bonds. A central sodium ion interacts with two thymines and contributes to the tetrad structure. (v) Completely disordered thymines which do not show any clear location in the crystal. The tetrads are stabilized by either Na+ or Tl+ ions. We show that by using MAD methods, Tl+ can be unambiguously located and distinguished from Na+. We can thus determine the preference for either ion in each ionic site of the structure under the conditions used by us.

Figure 1

Quadruplex columns in d(TGGGGT) crystals; left, the S1 structure in space group P1; right, the S2 structure in space group P2₁. Each quadruplex type is shown in a different color. A thymine tetrad is shown in black, with atoms as small spheres. It belongs to the red quadruplex. Ions are shown as yellow (Tl⁺) and pink (Na⁺) balls. Parallel columns are packed on the plane of the paper and form layers, which are stabilized by thymine interactions between neighbor columns. Layers parallel to the plane of the paper show no apparent contacts among themselves.

Figure 2

A view of one quadruplex in the S2 structure, with the anomalous fourier map superimposed. The thymine tetrad is at the top of the figure. Ions (Na⁺, Tl⁺) are shown as small spheres. The largest density peak is centered over TL1703 (occupancy 0.6) and the smaller peak over TL1704 (occupancy 0.4). Above and below are sodiums, and these do not show any density in the anomalous map.

Figure 3

(A) Comparison of the S1 (left) and S3 (right) structures. Thymines are shown in green. In S3, hydrated Ca²⁺ ions (in blue) occupy the central region of contact between G-tetrads, as described by Phillips and co-workers (2). In the S1 structure this region is occupied by thymines, four of which are well ordered in the grooves of the next quadruplex. The other four thymines are also found in the grooves of the other quadruplex, but are less well ordered. Note that in the S3 structure, the thymines occupy external positions and are poorly ordered, some sugars are even missing in the coordinate file. Divalent cations contribute to stabilize neighbor quadruplex columns: Ca²⁺ in S3 and Mg²⁺ (cyan) in S1. Hydration waters are not included. Monovalent ions are shown as yellow (Tl⁺) and pink (Na⁺) balls. Water molecules are red. (B and C) The two thymine triads (green) intercalated between the 3′-terminal G-tetrads of two quadruplexes. Sodium ions are shown in pink and water molecules in red. In (C), shown in projection, the upper G-tetrad has been removed. Hydrogen bonds (black dashed lines) are formed between the N3 and O4 atoms of two thymine pairs. The third thymine interacts through a water molecule. The drawing corresponds to S1, similar triads are present in S2 and S3.

Figure 4

Electron density omit map of the thymine tetrad in S2. The map, calculated using all the resolution range data, was contoured at 0.8 σ. Two of the thymines show higher disorder as it is apparent from the figure. The central ball corresponds to Na⁺. Hydrogen bonds and ionic interactions are shown as dashed lines.