Probing mechanism and transition state of RNA refolding.
Kinetics and the atomic detail of RNA refolding are only poorly understood. It has been proposed that conformations with transient base pairing interaction are populated during RNA refolding, but a detailed description of those states is lacking. By NMR and CD spectroscopy, we examined the refolding of a bistable RNA and the influence of urea, Mg(2+), and spermidine on its refolding kinetics. The bistable RNA serves as a model system and exhibits two almost equally stable ground-state conformations. We designed a photolabile caged RNA to selectively stabilize one of the two ground-state conformations and trigger RNA refolding by in situ light irradiation in the NMR spectrometer. We can show that the refolding kinetics of the bistable RNA is modulated by urea, Mg(2+), and spermidine by different mechanisms. From a statistical analysis based on elementary rate constants, we deduce the required number of base pairs that need to be destabilized during the refolding transition and propose a model for the transition state of the folding reaction.
Fürtig
Boris
B
Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.
Wenter
Philipp
P
Pitsch
Stefan
S
Schwalbe
Harald
H
D001483:Base Sequence; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry
D016337:RNA, Catalytic; D012313:RNA