Nanoscopic differences in free volume result in pressure-dependent changes in free energies which can therefore impact folding/unfolding stability of biomolecules. Although such effects are typically insignificant under ambient pressure conditions, they are crucially important for deep ocean marine life, where the hydraulic pressure can be on the kilobar scale. In this work, single molecule FRET spectroscopy is used to study the effects of pressure on both the kinetics and overall thermodynamics for folding/unfolding of the manganese riboswitch. Detailed pressure-dependent analysis of the conformational kinetics allows one to extract precision changes (σ ≲ 4-8 Å3) in free volumes not only between the fully folded/unfolded conformations but also with respect to the folding transition state of the manganese riboswitch. This permits first extraction of a novel \"reversible work\" free energy (PΔV) landscape, which reveals a monotonic increase in manganese riboswitch volume along the folding coordinate. Furthermore, such a tool permits exploration of pressure-dependent effects on both Mn2+ binding and riboswitch folding, which demonstrate that ligand attachment stabilizes the riboswitch under pressure by decreasing the volume increase upon folding (ΔΔV < 0). Such competition between ligand binding and pressure-induced denaturation dynamics could be of significant evolutionary advantage, compensating for a weakening in riboswitch tertiary structure with pressure-mediated ligand binding and promotion of folding response.

Conformational rearrangements are key to the function of riboswitches. These regulatory mRNA regions specifically bind to cellular metabolites using evolutionarily conserved sensing domains and modulate gene expression via adjacent downstream expression platforms, which carry gene expression signals. The regulation is achieved through the ligand-dependent formation of two alternative and mutually exclusive conformations involving the same RNA region. While X-ray crystallography cannot visualize dynamics of such dramatic conformational rearrangements, this method is pivotal to understand RNA-ligand interaction that stabilize the sensing domain and drive folding of the expression platform. X-ray crystallography can reveal local changes in RNA necessary for discriminating cognate and noncognate ligands. This chapter describes preparation of thiamine pyrophosphate riboswitch RNAs and its crystallization with different ligands, resulting in structures with local conformational changes in RNA. These structures can help to derive information on the dynamics of the RNA essential for specific binding to small molecules, with potential for using this information for developing designer riboswitch-ligand systems.

High-resolution mass spectrometry was used for the label-free, direct localization and relative quantification of CMC+ -modifications of a neomycin-sensing riboswitch aptamer domain in the absence and presence of the aminoglycoside ligands neomycin B, ribostamycin, and paromomycin. The chemical probing and MS data for the free riboswitch show high exposure to solvent of the uridine nucleobases U7, U8, U13, U14, U18 as part of the proposed internal and apical loops, but those of U10 and U21 as part of the proposed internal loop were found to be far less exposed than expected. Thus, our data are in better agreement with the proposed secondary structure of the riboswitch in complexes with aminoglycosides than with that of free RNA. For the riboswitch in complexes with neomycin B, ribostamycin, and paromomycin, we found highly similar CMC+ -modification patterns and excellent agreement with previous NMR studies. Differences between the chemical probing and MS data in the absence and presence of the aminoglycoside ligands were quantitative rather than qualitative (i. e., the same nucleobases were labeled, but to different extents) and can be rationalized by stabilization of both the proposed bulge and the apical loop by aminoglycoside binding. Our study shows that chemical probing and mass spectrometry can provide important structural information and complement other techniques such as NMR spectroscopy.

Crystallographic observation of structural changes in real time requires that those changes be uniform both spatially and temporally. A primary challenge with time-resolved ligand-mixing diffraction experiments is asynchrony caused by variable factors, such as efficiency of mixing, rate of diffusion, crystal size, and subsequently, conformational heterogeneity. One method of minimizing such variability is use of a photolabile caged ligand, which can fully saturate the crystal environment (spatially), and whose photoactivation can rapidly (temporally) trigger the reaction in a controlled manner. Our recently published results on a ligand-mixing experiment using time-resolved X-ray crystallography (TRX) with an X-ray free electron laser (XFEL) demonstrated that large conformational changes upon ligand binding resulted in a solid-to-solid phase transition (SSPT), while maintaining Bragg diffraction. Here we investigate this SSPT by polarized video microscopy (PVM) after light-triggered release of a photo-caged adenine (pcADE). In general, the mean transition times and transition widths of the SSPT were less dependent on crystal size than what was observed in previous PVM studies with direct ADE mixing. Instead, the photo-induced transition appears to be heavily influenced by the equilibrium between caged and uncaged ADE due to relatively low sample exposure and uncaging efficiency. Nevertheless, we successfully demonstrate a method for the characterization of phase transitions in RNA crystals that are inducible with a photocaged ligand. The transition data for three crystals of different sizes were then applied to kinetic analysis by fitting to the known four-state model associated with ligand-induced conformational changes, revealing an apparent concentration of uncaged ADE in crystal of 0.43-0.46 mM. These results provide further insight into approaches to study time-resolved ligand-induced conformational changes in crystals, and in particular, highlight the feasibility of triggering phase transitions using a light-inducible system. Developing such approaches may be paramount for the rapidly emerging field of time-resolved crystallography.

The spread of antibiotic-resistant bacteria represents a substantial health threat. Current antibiotics act on a few metabolic pathways, facilitating resistance. Consequently, novel regulatory inhibition mechanisms are necessary. Riboswitches represent promising targets for antibacterial drugs. Purine riboswitches are interesting, since they play essential roles in the genetic regulation of bacterial metabolism. Among these, class I (2\'-dG-I) and class II (2\'-dG-II) are two different 2\'-deoxyguanosine (2\'-dG) riboswitches involved in the control of deoxyguanosine metabolism. However, high affinity for nucleosides involves local or distal modifications around the ligand-binding pocket, depending on the class. Therefore, it is crucial to understand these riboswitches\' recognition mechanisms as antibiotic targets. In this work, we used a combination of computational biophysics approaches to investigate the structure, dynamics, and energy landscape of both 2\'-dG classes bound to the nucleoside ligands, 2\'-deoxyguanosine, and riboguanosine. Our results suggest that the stability and increased interactions in the three-way junction of 2\'-dG riboswitches were associated with a higher nucleoside ligand affinity. Also, structural changes in the 2\'-dG-II aptamers enable enhanced intramolecular communication. Overall, the 2\'-dG-II riboswitch might be a promising drug design target due to its ability to recognize both cognate and noncognate ligands.

The Hypr cGAMP signaling pathway was discovered via the function of the riboswitch. In this study, we show the development of a method for affinity capture followed by sequencing to identify non-coding RNA regions that bind nucleotide signals such as cGAMP. The RNAseq of affinity-captured cGAMP riboswitches from the Geobacter sulfurreducens transcriptome highlights general challenges that remain for this technique. Furthermore, by applying riboswitch reporters in vivo, we identify new growth conditions and transposon mutations that affect cGAMP levels in G. sulfurreducens. This work reveals an extensive regulatory network and supports a second functional cGAMP synthase gene in G. sulfurreducens. The activity of the second synthase was validated using riboswitch-based fluorescent biosensors, and is the first known example of an active enzyme with a variant GGDDF motif.

The thiamine pyrophosphate (TPP) riboswitch has emerged as the new target for designing new ligands for antibiotic purpose. Binding of the natural ligand TPP to the TPP riboswitch causes downregulation of the genes responsible for its biosynthesis. We have reported the role of π-stacking energy contributions to ligand binding with a TPP riboswitch. In conjunction with the docking study, the higher-level quantum chemical calculations performed with the wB97XD and Def2TZVPP basis set in the aqueous phase revealed that the optimum ring size is crucial to attain the effective binding efficiency of ligands with a TPP riboswitch. The π-stacking energy contributions observed for the ligands studied are largely similar; however, the cases studied with higher π-stacking energies with larger rings have a weaker ability to displace the radiolabeled thiamine from the riboswitch. The EDA and NCI analyses suggest the role of larger dispersive interactions in stabilizing the π-stacking rings. The contribution from hydrogen-bonding interactions of the hydrogen-bond donor groups on the A ring augments the binding affinity of the ligand. This study sheds light on various factors that contribute to the design of new ligands for efficient binding with a TPP riboswitch and inhibition of gene expression.

In this study, we provide experimental (Protein Data Bank (PDB) inspection) and theoretical (RI-MP2/def2-TZVP level of theory) evidence of the involvement of charge assisted chalcogen bonding (ChB) interactions in the recognition and folding mechanisms of S-adenosylmethionine (SAM) riboswitches. Concretely, an initial PDB search revealed several examples where ChBs between S-adenosyl methionine (SAM)/adenosyl selenomethionine (EEM) molecules and uracil (U) bases belonging to RNA take place. While these interactions are usually described as a merely Coulombic attraction between the positively charged S/Se group and RNA, theoretical calculations indicated that the σ holes of S and Se are involved. Moreover, computational models shed light on the strength and directionality properties of the interaction, which was also further characterized from a charge-density perspective using Bader\'s \"Atoms in Molecules\" (AIM) theory, Non-Covalent Interaction plot (NCIplot) visual index, and Natural Bonding Orbital (NBO) analyses. As far as our knowledge extends, this is the first time that ChBs in SAM-RNA complexes have been systematically analyzed, and we believe the results might be useful for scientists working in the field of RNA engineering and chemical biology as well as to increase the visibility of the interaction among the biological community.

Riboswitches are segments of noncoding RNA that bind with metabolites, resulting in a change in gene expression. To understand the molecular mechanism of gene regulation in a fluoride riboswitch, a base-pair opening dynamics study was performed with and without ligands using the Bacillus cereus fluoride riboswitch. We demonstrate that the structural stability of the fluoride riboswitch is caused by two steps depending on ligands. Upon binding of a magnesium ion, significant changes in a conformation of the riboswitch occur, resulting in the greatest increase in their stability and changes in dynamics by a fluoride ion. Examining hydrogen exchange dynamics through NMR spectroscopy, we reveal that the stabilization of the U45·A37 base-pair due to the binding of the fluoride ion, by changing the dynamics while maintaining the structure, results in transcription regulation. Our results demonstrate that the opening dynamics and stabilities of a fluoride riboswitch in different ion states are essential for the genetic switching mechanism.

Riboswitches are metabolite sensing aptamer domains present in non-coding regions in RNA and act as gene-regulating elements. Thiamine pyrophosphate (TPP) riboswitch is evolved as a new target for developing antibiotics against many pathogenic bacteria. The earlier reports suggest that the modification of the pyrophosphate group in the ligand molecule can enhance gene expression. In this work, we have examined the binding affinity and efficacy of TPP and two recently reported ligands, CH2-TPP, and CF2-TPP, using Well-tempered metadynamics (WT-MtD) simulations. The experimental in vitro assays show that both TPP and CH2-TPP repress the gene expression to the same extent. The calculated binding energies correlate well with the experimental study and show the same trend of binding affinity of ligands for the TPP riboswitch. The root mean square fluctuation profiles suggest that the CH2-TPP and TPP trigger higher fluctuations in P1 and L3 region, and such fluctuations in the P1 region is involved in the gene regulation process. The metal ion mediated contact of TPP ligand with pyrophosphate binding helix is found to be critical in the gene regulation process. The simulation results corroborate the experimental observations that the role of conformational changes occurring in different riboswitch regions upon ligand binding is essential to repress the gene expression process. This work sheds light on the subtle change in the ligand structure that can induce a more considerable impact on binding affinity and efficacy of ligands with riboswitch.