Monovalent and Divalent Promoted GAAA Tetraloop-Receptor Tertiary Interactions from Freely Diffusing Single-Molecule Studies

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 Mg(2+) 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 [Mg(2+)] 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 [Mg(2+)]-dependent docking decrease significantly for [Na(+)] = 25 mM vs. 125 mM, indicating Mg(2+) and Na(+) synergy in the RNA folding process.

Entropic origin of Mg 2+ -facilitated RNA folding

“…However, such a mechanism would require a [Mg 2þ ]-dependent decrease in the enthalpy for docking, which is not observed. This surprising result motivates us to investigate the role of ionic strength by reducing the background [NaCl] from 100 mM to 25 mM, at which charge repulsion should be amplified (21,29). We do in fact see the correct anticipated sign of this change, i.e., with reduction in [NaCl], ΔH ‡ dock slightly increases from weakly exothermic to thermoneutral at constant 1 mM Mg 2þ (Tables 1 and 2, Fig.…”

“…Unless otherwise specified, experiments are performed in 50 mM hemisodium Hepes buffer (pH 7.5 at 25°C) with 100 mM NaCl, 0.1 mM EDTA, and an oxygen scavenger of 60 nM protocatechuic acid, 5 mM protocatechuate-3,4-dioxygenase, and 2 mM Trolox (55) with the specified [Mg 2þ ] from added MgCl 2 . There is kinetic heterogeneity of the TL-R constructs under all conditions; experiments are performed on the approximately 70% population of actively docking species (16,28,29).…”

“…Emission from the donor (Cy3) and acceptor (Cy5) is spectrally separated for time-correlated single-photon counting detection using an inverted confocal microscope system (28,29) with temperature control (SI Text). Fluorescence trajectories of the donor and acceptor signal from single molecules are binned at 3-10 ms integration.…”

“…Linked by a flexible singlestranded poly(U) junction, the GAAA tetraloop facilely and specifically docks into its receptor, modulating the energy transfer efficiency (E FRET ) between the donor (Cy3) and acceptor (Cy5) fluorophores (16,28). E FRET is monitored by single-molecule confocal microscopy-calculated ratiometrically from the donor and acceptor emission intensities (Materials and Methods) (28,29). The RNA vacillates between two well-resolved states of high (docked) and low (undocked) E FRET , as seen by the superimposed fits in Fig.…”

“…1D and E) described by k dock or k undock represent the transition state for docking or undocking in a weighted average of k 1 and k 2 or k −1 and k −2 , respectively ( Fig. 2) (28,29,32). An increase in k dock and decrease in k undock with [Mg 2þ ] implies that k 1 < k 2 and k −2 < k −1 .…”