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T. maritima and B. subtilis are bacteria that inhabit significantly different thermal environments, ∼80 vs. ∼40°C, yet employ similar lysine riboswitches to aid in the transcriptional regulation of the genes involved in the synthesis and transport of amino acids. Despite notable differences in G-C basepair frequency and primary sequence, the aptamer moieties of each riboswitch have striking similarities in tertiary structure, with several conserved motifs and long-range interactions. To explore genetic adaptation in extreme thermal environments, we compare the kinetic and thermodynamic behaviors in T. maritima and B. subtilis lysine riboswitches via single-molecule fluorescence resonance energy transfer analysis. Kinetic studies reveal that riboswitch folding rates increase with lysine concentration while the unfolding rates are independent of lysine. This indicates that both riboswitches bind lysine through an induced-fit ("bind-then-fold") mechanism, with lysine binding necessarily preceding conformational changes. Temperature-dependent van't Hoff studies reveal qualitative similarities in the thermodynamic landscapes for both riboswitches in which progression from the open, lysine-unbound state to both transition states (‡) and closed, lysine-bound conformations is enthalpically favored yet entropically penalized, with comparisons of enthalpic and entropic contributions extrapolated to a common [K + ] = 100 mM in quantitative agreement. Finally, temperature-dependent Eyring analysis reveals the TMA and BSU riboswitches to have remarkably similar folding/unfolding rate constants when extrapolated to their respective (40 and 80°C) environmental temperatures. Such behavior suggests a shared strategy for ligand binding and aptamer conformational change in the two riboswitches, based on thermodynamic adaptations in number of G-C basepairs and/or modifications in tertiary structure that stabilize the ligand-unbound conformation to achieve biocompetence under both hyperthermophilic and mesothermophilic conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

G-Quadruplexes (G4s) are ubiquitous nucleic acid folding motifs that exhibit structural diversity that is dependent on cationic conditions. In this work, we exploit temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) to elucidate the kinetic and thermodynamic mechanisms by which monovalent cations (K + and Na + ) impact folding topologies for a simple G-quadruplex sequence (5'-GGG-(TAAGGG) 3 -3') with a three-state folding equilibrium. Kinetic measurements indicate that Na + and K + influence G4 formation in two distinctly different ways: the presence of Na + modestly enhances an antiparallel G4 topology through an induced fit (IF) mechanism with a low affinity ( K d = 228 ± 26 mM), while K + drives G4 into a parallel/hybrid topology via a conformational selection (CS) mechanism with much higher affinity ( K d = 1.9 ± 0.2 mM). Additionally, temperature-dependent studies of folding rate constants and equilibrium ratios reveal distinctly different thermodynamic driving forces behind G4 binding to K + (Δ H ° bind > 0, Δ S ° bind > 0) versus Na + (Δ H ° bind < 0, Δ S ° bind < 0), which further illuminates the diversity of the possible pathways for monovalent facilitation of G-quadruplex folding.

Riboswitches are highly structured RNA elements that regulate gene expressions by undergoing conformational changes in response to their cognate ligands. Such regulatory strategies are particularly prevalent among bacteria, which need to be evolutionarily responsive to thermal fluctuations in the surrounding environment, for example, generating extremophiles evolved to survive anomalously high or low temperatures. As a consequence, the response of such riboswitches to thermal stress becomes of considerable interest. In this study, the temperature-dependent folding kinetics and thermodynamics of the manganese riboswitch ( yybP-ykoY) is studied by single-molecule FRET spectroscopy under external thermal control. Surprisingly, the folding of the manganese riboswitch is found to be strongly promoted by temperature. Detailed thermodynamic analysis of the equilibrium and forward/reverse kinetic rate constants reveal folding to be a strongly endothermic process (Δ H 0 ) made feasible by an overall entropic lowering (- TΔ S 0 ) in free energy. This is in contrast to a more typical picture of RNA folding achieving a more compact, highly ordered state (Δ S 0 ) and clearly speaks to the significant role of solvent/cation reorganization in the folding thermodynamics. With the help of the transition-state theory, free energy landscapes for the manganese riboswitch are constructed from the temperature-dependent kinetic data, revealing two distinctive folding mechanisms promoted by Mg 2+ and Mn 2+ , respectively. It is speculated that this unconventional temperature dependence for folding of the manganese riboswitch may reflect evolution of bacterial gene regulation strategies to survive environments with large-temperature variations.

The effects of "molecular crowding" on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop-receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high-molecular-weight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG° ∼ -2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (Keq) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in Keq is dominated by a 35-fold increase in tetraloop-receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple "hard-sphere" treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG° ∼ -5 kcal/mol free-energy stabilization at excluded volume fractions of 30%.

Single-molecule fluorescence spectroscopy is a powerful technique that makes it possible to observe the conformational dynamics associated with biomolecular processes. The addition of precise temperature control to these experiments can yield valuable thermodynamic information about equilibrium and kinetic rate constants. To accomplish this, we have developed a microscopy technique based on infrared laser overtone/combination band absorption to heat small (≈10(-11) liter) volumes of water. Detailed experimental characterization of this technique reveals three major advantages over conventional stage heating methods: 1), a larger range of steady-state temperatures (20-100°C); 2), substantially superior spatial (≤20 μm) control; and 3), substantially superior temporal (≈1 ms) control. The flexibility and breadth of this spatial and temporally resolved laser-heating approach is demonstrated in single-molecule fluorescence assays designed to probe the dissociation of a 21 bp DNA duplex. These studies are used to support a kinetic model based on nucleic acid end fraying that describes dissociation for both short (<10 bp) and long (>10 bp) DNA duplexes. These measurements have been extended to explore temperature-dependent kinetics for the 21 bp construct, which permit determination of single-molecule activation enthalpies and entropies for DNA duplex dissociation., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cations have long been associated with formation of native RNA structure and are commonly thought to stabilize the formation of tertiary contacts by favorably interacting with the electrostatic potential of the RNA, giving rise to an "ion atmosphere". A significant amount of information regarding the thermodynamics of structural transitions in the presence of an ion atmosphere has accumulated and suggests stabilization is dominated by entropic terms. This work provides an analysis of how RNA-cation interactions affect the entropy and enthalpy associated with an RNA tertiary transition. Specifically, temperature-dependent single-molecule fluorescence resonance energy transfer studies have been exploited to determine the free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) of folding for an isolated tetraloop-receptor tertiary interaction as a function of Na(+) concentration. Somewhat unexpectedly, increasing the Na(+) concentration changes the folding enthalpy from a strongly exothermic process [e.g., ΔH° = -26(2) kcal/mol at 180 mM] to a weakly exothermic process [e.g., ΔH° = -4(1) kcal/mol at 630 mM]. As a direct corollary, it is the strong increase in folding entropy [Δ(ΔS°) > 0] that compensates for this loss of exothermicity for the achievement of more favorable folding [Δ(ΔG°) < 0] at higher Na(+) concentrations. In conjunction with corresponding measurements of the thermodynamics of the transition state barrier, these data provide a detailed description of the folding pathway associated with the GAAA tetraloop-receptor interaction as a function of Na(+) concentration. The results support a potentially universal mechanism for monovalent facilitated RNA folding, whereby an increasing monovalent concentration stabilizes tertiary structure by reducing the entropic penalty for folding.

The GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A(7) linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking {[Co(NH(3))(6)(3+)] << [Ca(2+)], [Mg(2+)], [Mn(2+)] << [Na(+)], [K(+)]}. Analysis of metal ion cooperativity yielded Hill coefficients of approximately 2 for Na(+)- or K(+)-dependent docking versus approximately 1 for the divalent ions and Co(NH(3))(6)(3+). Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U(7) and A(14) single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.

Single-molecule fluorescence microscopy techniques can be used in combination with micrometer length-scale temperature control and Förster resonance energy transfer (FRET) in order to gain detailed information about fundamental biophysical phenomena. In particular, this combination of techniques has helped foster the development of remarkable quantitative tools for studying both time- and temperature-dependent structural kinetics of biopolymers. Over the past decade, multiple research efforts have successfully incorporated precise spatial and temporal control of temperature into single-molecule FRET (smFRET)-based experiments, which have uncovered critical thermodynamic information on a wide range of biological systems such as conformational dynamics of nucleic acids. This review provides an overview of various temperature-dependent smFRET approaches from our laboratory and others, highlighting efforts in which such methods have been successfully applied to studies of single-molecule nucleic acid folding. [ABSTRACT FROM AUTHOR]

The ribonucleoprotein telomeraseis an RNA-dependent DNA polymerasethat catalyzes the repetitive addition of a short, species-specific,DNA sequence to the ends of linear eukaryotic chromosomes. The singleRNA component of telomerase contains both the template sequence forDNA synthesis and a functionally critical pseudoknot motif, whichcan also exist as a less stable hairpin. Here we use a minimal versionof the human telomerase RNA pseudoknot to study this hairpin–pseudoknotstructural equilibrium using temperature-controlled single-moleculefluorescence resonance energy transfer (smFRET) experiments. The ureadependence of these experiments aids in determination of the foldingkinetics and thermodynamics. The wild-type pseudoknot behavior iscompared and contrasted to a mutant pseudoknot sequence implicatedin a genetic disorder–dyskeratosis congenita. These findingsclearly identify that this 2nt noncomplementary mutation destabilizesthe folding of the wild-type pseudoknot by substantially reducingthe folding rate constant (≈ 400-fold) while only nominally increasingthe unfolding rate constant (≈ 5-fold).Furthermore, the urea dependence of the equilibrium and rate constantsis used to develop a free energy landscape for this unimolecular equilibriumand propose details about the structure of the transition state. Finally,the urea-dependent folding experiments provide valuable physical insightsinto the mechanism for destabilization of RNA pseudoknots by suchchemical denaturants. [ABSTRACT FROM AUTHOR]

Nearly two decades after Westhof and Michel first proposed that RNA tetraloops may interact with distal helices, tetraloop–receptor interactions have been recognized as ubiquitous elements of RNA tertiary structure. The unique architecture of GNRA tetraloops (N=any nucleotide, R=purine) enables interaction with a variety of receptors, e.g., helical minor grooves and asymmetric internal loops. The most common example of the latter is the GAAA tetraloop–11 nt tetraloop receptor motif. Biophysical characterization of this motif provided evidence for the modularity of RNA structure, with applications spanning improved crystallization methods to RNA tectonics. In this review, we identify and compare types of GNRA tetraloop–receptor interactions. Then we explore the abundance of structural, kinetic, and thermodynamic information on the frequently occurring and most widely studied GAAA tetraloop–11 nt receptor motif. Studies of this interaction have revealed powerful paradigms for structural assembly of RNA, as well as providing new insights into the roles of cations, transition states and protein chaperones in RNA folding pathways. However, further research will clearly be necessary to characterize other tetraloop–receptor and long-range tertiary binding interactions in detail – an important milestone in the quantitative prediction of free energy landscapes for RNA folding. [ABSTRACT FROM AUTHOR]

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, pKa, 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 (kdock), also affected by a strongly amino acid selective increase in the unfolding rate constant (kundock). To further elucidate the underlying thermodynamics, single-molecule equilibrium constants (Keq) 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. [ABSTRACT FROM AUTHOR]

Theinfluential role of monovalent and divalent metal cations infacilitating conformational transitions in both RNA and DNA has beena target of intense biophysical research efforts. However, organicneutrally charged cosolutes can also significantly alter nucleic acidconformational transitions. For example, highly soluble small moleculessuch as trimethylamine N-oxide (TMAO) and urea are occasionally utilizedby organisms to regulate cellular osmotic pressure. Ensemble studieshave revealed that these so-called osmolytes can substantially influencethe thermodynamics of nucleic acid conformational transitions. Inthe present work, we exploit single-molecule FRET (smFRET) techniquesto measure, for first time, the kinetic origins of these osmolyte-inducedchanges to the folding free energy. In particular, we focus on smFRETRNA and DNA constructs designed as model systems for secondary andtertiary structure formation. These findings reveal that TMAO preferentiallystabilizes both secondary and tertiary interactions by increasing kfoldand decreasing kunfold, whereas urea destabilizes both conformational transitions, resultingin the exact opposite shift in kinetic rate constants (i.e., decreasing kfoldand increasing kunfold). Complementary temperature-dependent smFRET experiments highlighta thermodynamic distinction between the two different mechanisms responsiblefor TMAO-facilitated conformational transitions, while only a singlemechanism is seen for the destabilizing osmolyte urea. Finally, theseresults are interpreted in the context of preferential interactionsbetween osmolytes, and the solvent accessible surface area (SASA)associated with the (i) nucleobase, (ii) sugar, and (iii) phosphategroups of nucleic acids in order to map out structural changes thatoccur during the conformational transitions. [ABSTRACT FROM AUTHOR]

Cations have long been associated with formation of native RNA structure and are commonly thought to stabilize the formation of tertiary contacts by favorably interacting with the electrostatic potential of the RNA, giving rise to an "ion atmosphere". A significant amount of information regarding the thermodynamics of structural transitions in the presence of an ion atmosphere has accumulated and suggests stabilization is dominated by entropic terms. This work provides an analysis of how RNA-cation interactions affect the entropy and enthalpy associated with an RNA tertiary transition. Specifically, temperature-dependent single-molecule fluorescence resonance energy transfer studies have been exploited to determine the free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) of folding for an isolated tetraloop-receptor tertiary interaction as a function of Na+ concentration. Somewhat unexpectedly, increasing the Na+ concentration changes the folding enthalpy from a strongly exothermic process [e.g., ΔH° = -26(2) kcal/mol at 180 mM] to a weakly exothermic process [e.g., ΔH° = - 4(l) kcal/mol at 630 mM]. As a direct corollary, it is the strong increase in folding entropy [Δ(ΔS°) > 0] that compensates for this loss of exothermicity for the achievement of more favorable folding [Δ(ΔG°) < 0] at higher Na+ concentrations. In conjunction with corresponding measurements of the thermodynamics of the transition state barrier, these data provide a detailed description of the folding pathway associated with the GAAA tetraloop-receptor interaction as a function of Na+ concentration. The results support a potentially universal mechanism for monovalent facilitated RNA folding, whereby an increasing monovalent concentration stabilizes tertiary structure by reducing the entropic penalty for folding. [ABSTRACT FROM AUTHOR]

RNA folding thermodynamics are crucial for structure prediction, which requires characterization of both enthalpic and entropic contributions of tertiary motifs to conformational stability. We explore the temperature dependence of RNA folding due to the ubiquitous GAAA tetraloop-receptor docking interaction, exploiting immobilized and freely diffusing single-molecule fluorescence resonance energy transfer (smFRET) methods. The equilibrium constant for intramolecular docking is obtained as a function of temperature (T = 21-47 °C), from which a van't Hoff analysis yields the enthalpy (ΔH°) and entropy (ΔS°) of docking. Tetraloop-receptor docking is significantly exothermic and entropically unfavorable in 1 mM MgCl2 and 100mM NaCl, with excellent agreement between immobilized (ΔH° = -17.4 ± 1.6 kcal/mol, and ΔS° = -56.2 ± 5.4 cal mol-1 K-1) and freely diffusing (AH° = -17.2 ± 1.6 kcal/mol, and AS° = -55.9 ± 5.2 cal mol-1 K-1) species. Kinetic heterogeneity in the tetraloop-receptor construct is unaffected over the temperature range investigated, indicating a large energy barrier for interconversion between the actively docking and nondocking subpopulations. Formation of the tetraloop-receptor interaction can account for ~60% of the ΔH° and ΔS° of P4-P6 domain folding in the Tetrahymena ribozyme, suggesting that it may act as a thermodynamic clamp for the domain. Comparison of the isolated tetraloop-receptor and other tertiary folding thermodynamics supports a theme that enthalpy- versus entropy-driven folding is determined by the number of hydrogen bonding and base stacking interactions. [ABSTRACT FROM AUTHOR]