Gene expression in bacteria is often regulated dynamically by conformational changes in a riboswitch upon ligand binding, a detailed understanding of which is very much in its infancy. For example, the manganese riboswitch is a widespread RNA motif that conformationally responds in regulating bacterial gene expression to micromolar levels of its eponymous ligand, Mn 2+ , but the mechanistic pathways are poorly understood. In this work, we quantitatively explore the dynamic folding behavior of the manganese riboswitch by single-molecule fluorescence resonance energy transfer spectroscopy as a function of cation/ligand conditions. From the detailed analysis of the kinetics, the Mn 2+ is shown to fold the riboswitch by a "bind-then-fold" (i.e., "induced-fit", IF) mechanism, whereby the ligand binds first and then promotes folding. On the other hand, the data also clearly reveal the presence of a folded yet ligand-free structure predominating due to the addition of physiological Mg 2+ to a nonselective metal ion binding site. Of particular kinetic interest, such a Mg 2+ "prefolded" conformation of the riboswitch is shown to exhibit a significantly increased affinity for Mn 2+ and further stabilization by subsequent binding of the ligand, thereby promoting efficient riboswitch folding by a "fold-then-bind" (i.e., "conformational selection", CS) mechanism. Our results not only demonstrate Mg 2+ -controlled switching between IF and CS riboswitch folding pathways but also suggest a novel heterotropic allosteric control in the manganese riboswitch activity co-regulated by Mg 2+ binding.

In light of the current models for an early RNA-based universe, the potential influence of simple amino acids on tertiary folding of ribozymal RNA into biochemically competent structures is speculated to be of significant evolutionary importance. In the present work, the folding–unfolding kinetics of a ubiquitous tertiary interaction motif, the GAAA tetraloop–tetraloop receptor (TL–TLR), is investigated by single-molecule fluorescence resonance energy transfer spectroscopy in the presence of natural amino acids both with (e.g., lysine, arginine) and without (e.g., glycine) protonated side chain residues. By way of control, we also investigate the effects of a special amino acid (e.g., proline) and amino acid mimetic (e.g., betaine) that contain secondary or quaternary amine groups rather than a primary amine group. This combination permits systematic study of amino acid induced (or amino acid like) RNA folding dynamics as a function of side chain complexity, pK a, charge state, and amine group content. Most importantly, each of the naturally occurring amino acids is found to destabilize the TL–TLR tertiary folding equilibrium, the kinetic origin of which is dominated by a decrease in the folding rate constant (k dock), also affected by a strongly amino acid selective increase in the unfolding rate constant (k undock). To further elucidate the underlying thermodynamics, single-molecule equilibrium constants (K eq) for TL–TLR folding have been probed as a function of temperature, which reveal an amino acid dependent decrease in both overall exothermicity (ΔΔH° > 0) and entropic cost (−TΔΔS° < 0) for the overall folding process. Temperature-dependent studies on the folding/unfolding kinetic rate constants reveal analogous amino acid specific changes in both enthalpy (ΔΔH ⧧) and entropy (ΔΔS ⧧) for accessing the transition state barrier. The maximum destabilization of the TL–TLR tertiary interaction is observed for arginine, which is consistent with early studies of arginine and guanidine ion-inhibited self-splicing kinetics for the full Tetrahymena ribozyme [YarusM.ChristianE. L. Yarus, M. Christian, E. L. Nature1989342349350; YarusM. Yarus, M. Science198824017511758].

The cell is an extremely crowded environment, which is known to have a profound impact on the thermodynamics, functionality, and conformational stability of biomolecules. Speculations from recent theoretical molecular dynamics studies suggest an intriguing size dependence to such purely entropic crowding effects, whereby small molecular weight crowders under constant enthalpy conditions are more effective than larger crowders on a per volume basis. If experimentally confirmed, this would be profoundly significant, as the cellular cytoplasm is also quite concentrated in smaller molecular weight solutes such as inorganic ions, amino acids, and various metabolites. The challenge is to perform such studies isolating entropic effects under isoenthalpic conditions. In this work, we first present results from single-molecule FRET spectroscopy (smFRET) on the molecular size-dependent crowding stabilization of a simple RNA tertiary motif (the GAAA tetraloop–tetraloop receptor), indeed providing evidence in support of the surprising notion in the crowding literature that “smaller is better.” Specifically, systematic smFRET studies as a function of crowder solute size reveal that smaller molecules both significantly increase the RNA tertiary folding rate and, yet, simultaneously decrease the unfolding rate, predicting strongly size-dependent stabilization of RNA tertiary structures under crowded cellular conditions. The size dependence of these effects has been explored via systematic variation of crowder size over a broad range of molecular weights (90–3000 amu). Furthermore, corresponding temperature dependent studies indicate the systematic changes in the folding equilibrium to be predominantly entropic in origin, i.e., consistent with a fundamental picture of entropic molecular crowding without additional enthalpic interactions. Most importantly, all trends in the single-molecule crowding data can be quantitatively recapitulated by a simple analytic depletion force model, whereby excluded volume interactions represent the major thermodynamic driving force toward folding. Our study, thus, not only provides experimental evidence and theoretical support for small molecule crowding but also predicts further enhancement of crowding effects for even smaller molecules on a per volume basis.