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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... Full description

Journal Title: Nucleic acids research 2007, Vol.35(6), pp.2047-2059
Main Author: Kobitski, Andrei Yu
Other Authors: Nierth, Alexander , Helm, Mark , Jäschke, Andres , Nienhaus, G Ulrich
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: E-ISSN: 1362-4962
Link: http://search.proquest.com/docview/70416051/?pq-origsite=primo
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title: Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis.
format: Article
creator:
  • Kobitski, Andrei Yu
  • Nierth, Alexander
  • Helm, Mark
  • Jäschke, Andres
  • Nienhaus, G Ulrich
subjects:
  • Cations, Divalent–Chemistry
  • Fluorescence Resonance Energy Transfer–Chemistry
  • Magnesium–Chemistry
  • Models, Molecular–Chemistry
  • Nucleic Acid Conformation–Chemistry
  • RNA, Catalytic–Chemistry
  • Thermodynamics–Chemistry
  • Cations, Divalent
  • RNA, Catalytic
  • Magnesium
ispartof: Nucleic acids research, 2007, Vol.35(6), pp.2047-2059
description: 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.
language: eng
source:
identifier: E-ISSN: 1362-4962
fulltext: fulltext
issn:
  • 13624962
  • 1362-4962
url: Link


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titleMg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis.
creatorKobitski, Andrei Yu ; Nierth, Alexander ; Helm, Mark ; Jäschke, Andres ; Nienhaus, G Ulrich
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identifierE-ISSN: 1362-4962
subjectCations, Divalent–Chemistry ; Fluorescence Resonance Energy Transfer–Chemistry ; Magnesium–Chemistry ; Models, Molecular–Chemistry ; Nucleic Acid Conformation–Chemistry ; RNA, Catalytic–Chemistry ; Thermodynamics–Chemistry ; Cations, Divalent ; RNA, Catalytic ; Magnesium
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descriptionHere, 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.
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