Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis.
Here, we report a single-molecule fluorescence resonance energy transfer (FRET) study of a Diels-Alderase (DAse) ribozyme, a 49-mer RNA with true catalytic properties. The DAse ribozyme was labeled with Cy3 and Cy5 as a FRET pair of dyes to observe intramolecular folding, which is a prerequisite for its recognition and turnover of two organic substrate molecules. FRET efficiency histograms and kinetic data were taken on a large number of surface-immobilized ribozyme molecules as a function of the Mg(2+) concentration in the buffer solution. From these data, three separate states of the DAse ribozyme can be distinguished, the unfolded (U), intermediate (I) and folded (F) states. A thermodynamic model was developed to quantitatively analyze the dependence of these states on the Mg(2+) concentration. The FRET data also provide information on structural properties. The I state shows a strongly cooperative compaction with increasing Mg(2+) concentration that arises from association with several Mg(2+) ions. This transition is followed by a second Mg(2+)-dependent cooperative transition to the F state. The observation of conformational heterogeneity and continuous fluctuations between the I and F states on the approximately 100 ms timescale offers insight into the folding dynamics of this ribozyme.
Kobitski
Andrei Yu
AY
Institute of Biophysics, University of Ulm, 89069 Ulm, Germany.
Nierth
Alexander
A
Helm
Mark
M
Jäschke
Andres
A
Nienhaus
G Ulrich
GU
D002413:Cations, Divalent / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D008274:Magnesium / Q000737:chemistry*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry*; D013816:Thermodynamics
D002413:Cations, Divalent; D016337:RNA, Catalytic; D008274:Magnesium