Naturally occurring RNA pseudoknots fold into many topologies, yet their formation is poorly understood. Herein, by using high-resolution single-molecule force spectroscopy, the folding pathways of the H-type pseudoknot found in the preQ1-riboswitch in B. subtilis were investigated By holding a single riboswitch RNA molecule in the optical-trap, the structural rearrangements as the end-to-end distance change along the pulling direction, x at a force, F were followed. The data reveal a multistate folding, wherein the intermediate hairpin undergoes a unidirectional conformational switching in the presence of ligand to form the pseudoknot receptor. Specifically-designed mutant RNAs resisted the switching mechanism and resulted in a significantly reduced pseudoknot population (4.5 percent) compared to the wild-type (100 percent). The free-energy landscape highlighted two kinetic barriers (DELTAG plus or minus ) that interrupt the folding pathway. By coupling the exothermic ligand-binding reaction (DELTAGbinding=-16 kT) to the folding events, the nascent transcript ensures successful barrier crossing,...

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