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Nanomanipulation of Single RNA Molecules by Optical Tweezers
06:59

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Published on: August 20, 2014

A large collapsed-state RNA can exhibit simple exponential single-molecule dynamics.

Glenna J Smith1, Kang Taek Lee, Xiaohui Qu

  • 1Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA.

Journal of Molecular Biology
|April 12, 2008
PubMed
Summary

Large RNA folding unexpectedly shows simple kinetics, revealing discrete states even at low magnesium. Critical interactions stabilize conformations, simplifying dynamics in the Bacillus stearothermophilus RNase P RNA catalytic domain.

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Area of Science:

  • Biochemistry and Molecular Biology
  • Structural Biology
  • RNA Biophysics

Background:

  • Large RNA folding is complex, often leading to diverse collapsed structures before achieving a unique functional state.
  • Non-exponential folding kinetics are typically expected for large RNAs not in their native state due to structural heterogeneity.
  • The catalytic domain of RNase P RNA from Bacillus stearothermophilus (C(thermo)) is a well-studied model for RNA folding.

Purpose of the Study:

  • To investigate the conformational dynamics of the collapsed-state region of the C(thermo) RNase P RNA free energy landscape.
  • To understand RNA folding kinetics at low ionic strength (magnesium ion concentration) where the native state is not achieved.

Main Methods:

  • Single-molecule fluorescence resonance energy transfer (smFRET) was employed to study equilibrium conformational fluctuations.
  • Measurements were performed on an ensemble of 300 single C(thermo) RNA molecules at various magnesium concentrations.
  • Analysis of dwell time distributions was used to infer the number of conformational states and their kinetics.

Main Results:

  • Contrary to expectations, simple exponential kinetics were observed, indicating a limited number of discrete conformational states.
  • Dwell time distributions consistently fit a double exponential model, suggesting a four-state system for RNA conformational changes.
  • Well-defined conformations were observed on the second timescale even at low magnesium concentrations (<0.1 mM).

Conclusions:

  • Even at low ionic strength, critical interactions can create deep energy wells, leading to simplified folding kinetics for large RNAs.
  • The observed discrete states and simple kinetics challenge the assumption of highly inhomogeneous dynamics in partially folded large RNAs.
  • These findings highlight the role of specific interactions in stabilizing key conformations, overriding the potential for vast structural diversity.