Mg²⁺-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules

Fluorescence correlation spectroscopy (FCS) of fluorescence resonant energy transfer (FRET) on immobilized individual fluorophores was used to study the Mg²⁺-facilitated conformational change of an RNA three-helix junction, a structural element that initiates the folding of the 30S ribosomal subunit. Transitions of the RNA junction between open and folded conformations resulted in fluctuations in fluorescence by FRET. Fluorescence fluctuations occurring between two FRET states on the millisecond time scale were found to be dependent on Mg²⁺ and Na⁺ concentrations. Correlation functions of the fluctuations were used to determine transition rates between the two conformations as a function of Mg²⁺ or Na⁺ concentration. Both the opening and folding rates were found to vary with changing salt conditions. Assuming specific binding of divalent ions to RNA, the Mg²⁺ dependence of the observed rates cannot be explained by conformational change induced by Mg²⁺ binding/unbinding, but is consistent with a model in which the intrinsic conformational change of the RNA junction is altered by uptake of Mg²⁺ ion(s). This version of FCS/FRET on immobilized single molecules is demonstrated to be a powerful technique in the study of conformational dynamics of biomolecules over time scales ranging from microseconds to seconds.