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Abstract

Riboswitch RNAs regulate gene expression by conformational changes induced by environmental conditions and specific ligand binding. The guanidine-II riboswitch is proposed to bind the small molecule guanidinium and to subsequently form a kissing loop interaction between the P1 and P2 hairpins. While an interaction was shown for isolated hairpins in crystallization and electron paramagnetic resonance experiments, an intrastrand kissing loop formation has not been demonstrated. Here, we report the first evidence of this interaction in cis in a ligand and Mg²⁺ dependent manner. Using single-molecule FRET spectroscopy and detailed structural information from coarse-grained simulations, we observe and characterize three interconvertible states representing an open and kissing loop conformation as well as a novel Mg²⁺ dependent state for the guanidine-II riboswitch from E. coli. The results further substantiate the proposed switching mechanism and provide detailed insight into the regulation mechanism for the guanidine-II riboswitch class. Combining single molecule experiments and coarse-grained simulations therefore provides a promising perspective in resolving the conformational changes induced by environmental conditions and to yield molecular insights into RNA regulation.