High-Precision FRET to Analyze the Architecture and Heterogeneity of RNA Junctions

Abstract

Like for many other non-coding RNAs, helical four-way and three-way junctions (4WJs and 3WJs) are an essential structural motif of the for functional RNA structures. Using FRET restrained high-precision structural modeling as a hybrid tool we resolve the structures of three coexisting conformers of a fully Watson-Crick base paired RNA4WJ based on the hairpin ribozyme. 51 different FRET-pairs were measured using single-molecule multi-parameter fluorescence detection (smMFD). For each dataset, the single-molecule approach allowed for the simultaneous extraction of three distances (and their corresponding errors) belonging to one major FRET state and two minor states. Distinct Mg2+-affinities were used for the assignment of the two minor states to the corresponding conformers. Rigid body models for the major and both minor conformers were obtained by docking rigid ds A-RNA helices explicitly taking into account dye position distributions. The three rigid body models were refined by all-atom MD simulations and coarse-grained RNA folding using FRET-restraints. A cluster analysis gives confidence levels for the proposed ensemble of models, and the precision was assessed via bootstrapping. The achieved precisions are significantly better than the uncertainty of the dye position with respect to the macromolecule. The structure of the 4WJ was compared with FRET restrained structures of related RNA 3WJs, where one stem was removed. The types of 3WJs were studied: (I) without bulges, (II) with a small bulge (two unpaired nucleotides) and (III) with larger bulge (5 unpaired nucleotides). In conclusion the overall geometry of the RNA helices depends drastically on the junction type.[1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010)[2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011)[3] Kalinin, S. et al. Nat. Methods in press