Monovalent and divalent promoted GAAA tetraloop-receptor tertiary interactions from freely diffusing single-molecule studies |
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Authors: | Fiore Julie L Hodak Jose H Piestert Oliver Downey Christopher D Nesbitt David J |
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Institution: | * JILA, National Institute of Standards and Technology and University of Colorado, Boulder, Colorado 80309 † Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309 |
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Abstract: | 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 KD 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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