Single-molecule studies of DNA and RNA four-way junctions

Abstract

Branched helical junctions are common in nucleic acids. In DNA, the four-way junction (Holliday junction) is an essential intermediate in homologous recombination and is a highly dynamic structure, capable of stacking conformer transitions and branch migration. Our single-molecule fluorescence studies provide unique insight into the energy landscape of Holliday junctions by visualizing these processes directly. In the hairpin ribozyme, an RNA four-way junction is an important structural element that enhances active-site formation by several orders of magnitude. Our single-molecule studies suggest a plausible mechanism for how the junction achieves this remarkable feat; the structural dynamics of the four-way junction bring about frequent contacts between the loops that are needed to form the active site. The most definitive evidence for this is the observation of three-state folding in single-hairpin ribozymes, the intermediate state of which is populated due to the intrinsic properties of the junction.

DEAD-box-protein-assisted RNA Structure Conversion Towards and Against Thermodynamic Equilibrium Values

“…Our data do not indicate whether Ded1 (without ATP) accelerates the putative branch migration process, but we note that branch migrations can occur spontaneously, as has been observed in other systems. 22,23 Irrespective of the actual mode of transition from the tripartite species to the final RNA structure, our results suggest that RNA structure conversions can be facilitated through stabilization of inherently unstable RNA species. While stabilization of the weak RNA-RNA interactions by proteins may be important throughout RNA metabolism, 1,2,[24][25][26] such structure stabilization has never been observed directly for a DEAD-box protein.…”

Towards understanding the catalytic core structure of the spliceosome

“…This observation lends further support to the hypothesis that the spliceosome and group II introns share a common molecular ancestor. Secondly, four-way junctions form coaxial helical stacks that could juxtapose catalytically essential elements, as observed in the hairpin ribozyme [23]. We utilized the crystal structure of a hairpin ribozyme four-way junction [24] to model such an interaction [20], and the resulting model was satisfying in that it predicted a close proximity between the U6 ISL metal-binding site and the intron-binding regions of U2 and U6 ( Figure 2B).…”

Development and Applications of Fluorescent Oligonucleotides

“…Dynamic characteristics of NAs can be studied using this technique. Recent examples including hairpin ribozymes, Holliday junctions, Gquadruplexes, Rep helicase and reverse transcriptases have been reviewed [36,280].…”