Journal
BIOPHYSICAL JOURNAL
Volume 95, Issue 8, Pages 3892-3905Publisher
CELL PRESS
DOI: 10.1529/biophysj.108.134346
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Funding
- National Science Foundation
- W. M. Keck Foundation
- Optical Science and Engineering Program National Science Foundation-Integrative Graduate Education and Research Traineeship
- University of Colorado Biophysics Training [T32 GM-065103]
- National Institutes of Health Biophysics Training [GM-065103]
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Proper assembly of RNA into catalytically active three-dimensional structures requires multiple tertiary binding interactions, individual characterization of which is crucial to a detailed understanding of global RNA folding. This work focuses on single-molecule fluorescence studies of freely diffusing RNA constructs that isolate the GAAA tetraloop-receptor tertiary interaction. Freely diffusing conformational dynamics are explored as a function of Mg2(+) and Na+ concentration, both of which promote facile docking, but with 500-fold different affinities. Systematic shifts in mean fluorescence resonance energy transfer efficiency values and line widths with increasing [Na+] are observed for the undocked species and can be interpreted with a Debye model in terms of electrostatic relaxation and increased flexibility in the RNA. Furthermore, we identify a 34 +/- 2% fraction of freely diffusing RNA constructs remaining undocked even at saturating [Mg2+] levels, which agrees quantitatively with the 32 +/- 1% fraction previously reported for immobilized constructs. This verifies that the kinetic heterogeneity observed in the docking rates is not the result of surface tethering. Finally, the K-D value and Hill coefficient for [Mg2+]-dependent docking decrease significantly for [Na+] = 25 mM vs. 125 mM, indicating Mg2+ and Na+ synergy in the RNA folding process.
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