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Nucleic Acids Research Advance Access originally published online on March 7, 2007
Nucleic Acids Research 2007 35(6):2047-2059; doi:10.1093/nar/gkm072
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Nucleic Acids Research, 2007, Vol. 35, No. 6 2047-2059
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis

Andrei Yu. Kobitski1, Alexander Nierth2, Mark Helm2, Andres Jäschke2 and G. Ulrich Nienhaus1,3,*

1Institute of Biophysics, University of Ulm, 89069 Ulm, Germany, 2Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, 69120 Heidelberg, Germany and 3Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

*To whom correspondence should be addressed. Tel: +1-49-731-50-23050; Fax: +1-49-731-50-23059; Email: uli{at}uiuc.edu

Received October 1, 2006. Revised January 23, 2007. Accepted January 24, 2007.

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 Mg2+ 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 Mg2+ concentration. The FRET data also provide information on structural properties. The I state shows a strongly cooperative compaction with increasing Mg2+ concentration that arises from association with several Mg2+ ions. This transition is followed by a second Mg2+-dependent cooperative transition to the F state. The observation of conformational heterogeneity and continuous fluctuations between the I and F states on the ~100 ms timescale offers insight into the folding dynamics of this ribozyme.


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