Molecular structure of a stacked Holliday junction, in which the four arms stack into two double-helical domains. Note how the blue and red strands remain roughly helical, while the green and yellow strands cross over between the two domains.
Molecular structure of an unstacked Holliday junction. This conformation lacks base stacking
between the double-helical domains, and is stable only in solutions lacking divalent metal ions such as Mg2+
. From 3CRX 3CRX
Schematic diagrams of the three base-stacking conformational isomers
of the Holliday junction. The two stacked conformers differ in which sets of two arms are bound by coaxial stacking
: at left, the stacks are red–blue and cyan–magenta, while at right the stacks are red–cyan and blue–magenta. The bases nearest to the junction point determine which stacked isomer dominates.
Holliday junctions may exist in a variety of conformational isomers with different patterns of coaxial stacking between the four double-helical arms. Coaxial stacking is the tendency of nucleic acid blunt ends to bind to each other, by interactions between the exposed bases. There are three possible conformers: an unstacked form and two stacked forms. The unstacked form dominates in the absence of divalent cations such as Mg2+, because of electrostatic repulsion between the negatively charged backbones of the strands. In the presence of at least about 0.1 mM Mg2+, the electrostatic repulsion is counteracted and the stacked structures predominate. As of 2000, it was not known with certainty whether the electrostatic shielding was the result of site-specific binding of cations to the junction, or the presence of a diffuse collection of the ions in solution.
The unstacked form is a nearly square planar, extended conformation. On the other hand, the stacked conformers have two continuous double-helical domains separated by an angle of about 60° in a right-handed direction. Two of the four strands stay roughly helical, remaining within each of the two double-helical domains, while the other two cross between the two domains in an antiparallel fashion.
The two possible stacked forms differ in which pairs of the arms are stacked with each other; which of the two dominates is highly dependent on the base sequences nearest to the junction. Some sequences result in an equilibrium between the two conformers, while others strongly prefer a single conformer. In particular, junctions containing the sequence A-CC bridging the junction point appear to strongly prefer the conformer that allows a hydrogen bond to form between the second cytosine and one of the phosphates at the junction point. While most studies have focused on the identities of the four bases nearest to the junction on each arm, it is evident that bases farther out can also affect the observed stacking conformations.
In junctions with symmetrical sequences, the branchpoint is mobile and can migrate in a random walk process. The rate of branch migration varies dramatically with ion concentration, with single-step times increasing from 0.3−0.4 ms with no ions to 270−300 ms with 10 mM Mg2+. The change in rate is correlated with the formation of the stacked versus the unstacked structures.
Holliday junctions with a nick, or break in one of the strands, at the junction point adopt a perpendicular orientation, and always prefer the stacking conformer that places the nick on a crossover strand rather than a helical strand.
RNA Holliday junctions assume an antiparallel stacked conformation at high magnesium concentrations, a perpendicular stacked conformation at moderate concentrations, and rotate into a parallel stacked conformation at low concentrations, while even small calcium ion concentrations favor the antiparallel conformer.