Topology of Large RNA Junctions Explored by High-Precision FRET

Abstract

Non-protein coding RNAs perform essential functions in living organisms. They commonly exist as dynamic ensembles of conformational states. While many structurally known RNAs are trapped in one or a few conformations by interactions with proteins or tertiary contacts between stems, bulges, or loops the knowledge of equilibrium structures, conformational space, and tertiary conformational changes of large RNAs not being restrained by external or tertiary interactions is still very limited.Helical four-way and three-way junctions (4WJs and 3WJs) are an essential structural motif of the for functional RNA structures. Here we explore the topology of a set of a 4WJ and related 3WJs related to the hairpin ribozyme by measuring more than 250 different FRET-pairs using single-molecule multi-parameter fluorescence detection [1]. Using FRET restrained high-precision structural modeling combined with full atom MD simulations as a hybrid tool [2,3], we resolve the structures of three coexisting conformers of a fully Watson-Crick base paired RNA4WJ. By a suitable choice of the number of bases in the bulges the helices arrangements of the corresponding 3WJs can span a huge conformational space which is necessary for the stem communication in functional RNAs.[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 9, 1218-1225 (2012)