The Second Class of Tetrahydrofolate (THF-II) Riboswitches Recognizes the Tetrahydrofolic Acid Ligand via Local Conformation Changes

Riboswitches are regulatory noncoding RNAs found in bacteria, fungi and plants, that modulate gene expressions through structural changes in response to ligand binding. Understanding how ligands interact with riboswitches in solution can shed light on the molecular mechanisms of this ancient regulators. Previous studies showed that riboswitches undergo global conformation changes in response to ligand binding to relay information. Here, we report conformation switching models of the recently discovered tetrahydrofolic acid-responsive second class of tetrahydrofolate (THF-II) riboswitches in response to ligand binding. Using a combination of selective 2′-hydroxyl acylation, analyzed by primer extension (SHAPE) assay, 3D modeling and small-angle X-ray scattering (SAXS), we found that the ligand specifically recognizes and reshapes the THF-II riboswitch loop regions, but does not affect the stability of the P3 helix. Our results show that the THF-II riboswitch undergoes only local conformation changes in response to ligand binding, rearranging the Loop1-P3-Loop2 region and rotating Loop1 from a ~120° angle to a ~75° angle. This distinct conformation changes suggest a unique regulatory mechanism of the THF-II riboswitch, previously unseen in other riboswitches. Our findings may contribute to the fields of RNA sensors and drug design.     

Characterization of Structure and Dynamics of the Guanidine-II Riboswitch from Escherichia coli by NMR Spectroscopy and Small-Angle X-ray Scattering (SAXS)

Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg²⁺ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg²⁺ and ligand binding.     

Incorporation of a FRET Pair into a Riboswitch RNA to Measure Mg2+ Concentration and RNA Conformational Change in Cell

Riboswitches are natural biosensors that can regulate gene expression by sensing small molecules. Knowledge of the structural dynamics of riboswitches is crucial to elucidate their regulatory mechanism and develop RNA biosensors. In this work, we incorporated the fluorophore, Cy3, and its quencher, TQ3, into a full-length adenine riboswitch RNA and its isolated aptamer domain to monitor the dynamics of the RNAs in vitro and in cell. The adenine riboswitch was sensitive to Mg²⁺ concentrations and could be used as a biosensor to measure cellular Mg²⁺ concentrations. Additionally, the TQ3/Cy3-labeled adenine riboswitch yielded a Mg²⁺ concentration that was similar to that measured using a commercial assay kit. Furthermore, the fluorescence response to the adenine of the TQ3/Cy3-labeled riboswitch RNA was applied to determine the proportions of multiple RNA conformational changes in cells. The strategy developed in this work can be used to probe the dynamics of other RNAs in cells and may facilitate the developments of RNA biosensors, drugs and engineering.     

The corrin moiety of coenzyme B12 is the determinant for switching the btuB riboswitch of E. coli

Riboswitches are regulatory elements in the 5'-untranslated region (5'-UTR) of bacterial mRNAs that bind certain metabolites with high specificity and affinity. The 202 nucleotide (nt)-long btuB riboswitch RNA of E. coli interacts specifically with coenzyme B12 and its derivatives thereby leading to changes in the RNA structure and hence to an altered expression of the downstream btuB gene. We report the investigations of the rearrangement of the three-dimensional structure of the btuB riboswitch upon binding to four different B12 derivatives: coenzyme B12, vitamin B12, adenosyl factor A and adenosyl-cobinamide. In-line probing experiments have shown that the corrin ring plays the crucial role in switching the three-dimensional riboswitch structure. Instead, the apical ligands influence only the binding affinity of the B12 derivative to the btuB riboswitch.     

Analysis of metal ion dependence in glmS ribozyme self-cleavage and coenzyme binding

The bacterial glmS ribozyme is mechanistically unique among both riboswitches and RNA catalysts. Its self-cleavage activity is the basis of riboswitch regulation of glucosamine-6-phosphate (GlcN6P) production, and catalysis requires GlcN6P as a coenzyme. Previous work has shown that the coenzyme amine of GlcN6P is essential for glmS ribozyme self-cleavage, as is its protonation state. Metal ions are also essential within the glmS ribozyme core for both structure and function of the ribozyme. Although metal ions do not directly promote catalysis, we show that metal ion identity and the varying physicochemical properties of metal ions have an impact on the rate of glmS ribozyme self-cleavage. Specifically, these studies demonstrate that metal ion identity influences the overall apparent pK(a) of ribozyme self-cleavage, and metal ion binding largely reflects phosphate oxygen affinity. Results suggest that metal ions take alternative roles in supporting the mechanism of catalysis.     

The Cytoskeletal Protein Zyxin Inhibits Retinoic Acid Signaling by Destabilizing the Maternal mRNA of the RXRγ Nuclear Receptor

Zyxin is an LIM-domain-containing protein that regulates the assembly of F-actin filaments in cell contacts. Additionally, as a result of mechanical stress, Zyxin can enter nuclei and regulate gene expression. Previously, we found that Zyxin could affect mRNA stability of the maternally derived stemness factors of Pou5f3 family in Xenopus laevis embryos through binding to Y-box factor1. In the present work, we demonstrate that Zyxin can also affect mRNA stability of the maternally derived retinoid receptor Rxrγ through the same mechanism. Moreover, we confirmed the functional link between Zyxin and Rxrγ-dependent gene expression. As a result, Zyxin appears to play an essential role in the regulation of the retinoic acid signal pathway during early embryonic development. Besides, our research indicates that the mechanism based on the mRNA destabilization by Zyxin may take part in the control of the expression of a fairly wide range of maternal genes.     

Differential Scanning Fluorimetry for Monitoring RNA Stability

Cellular RNA function is closely linked to RNA structure. It is therefore imperative to develop methods that report on structural stability of RNA and how it is modulated by binding of ions, other osmolytes, and RNA‐binding ligands. Here, we present a novel method to analyze the stability of virtually any structured RNA in a highly parallel fashion. This method can easily determine the influence of various additives on RNA stability, and even characterize ligand‐induced stabilization of riboswitch RNA. Current approaches to assess RNA stability include thermal melting profiles (absorption or circular dichroism) and differential scanning calorimetry. These techniques, however, require a substantial amount of material and cannot be significantly parallelized. Current fluorescence spectroscopic methods rely on intercalating dyes, which alter the stability of RNA. We employ the commercial fluorescent dye RiboGreen, which discriminates between single‐stranded (or unstructured regions) and double‐stranded RNA. Binding leads to an increase in fluorescence quantum yield, and thus reports structural changes by a change in fluorescence intensity.     

Phosphate-group recognition by the aptamer domain of the thiamine pyrophosphate sensing riboswitch

Riboswitches are highly structured RNA elements that control gene expression by binding directly to small metabolite molecules. Remarkably, many of these metabolites contain negatively charged phosphate groups that contribute significantly to the binding affinity. An example is the thiamine pyrophosphate-sensing riboswitch in the 5'-untranslated region of the E. coli thiM mRNA. This riboswitch binds, in order of decreasing affinity, to thiamine pyrophosphate (TPP), thiamine monophosphate (TMP), and thiamine, which contain two, one, and no phosphate groups, respectively. We examined the binding of TPP and TMP to this riboswitch by using (31)P NMR spectroscopy. Chemical-shift changes were observed for the alpha- and beta-phosphate group of TPP and the phosphate group of TMP upon RNA binding; this indicates that they are in close contact with the RNA. Titration experiments with paramagnetic Mn(2+) ions revealed strong line-broadening effects for both (31)P signals of the bound TPP; this indicates a Mg(2+) binding site in close proximity and suggests that the phosphate group(s) of the ligand is/are recognized in a magnesium ion-mediated manner by the RNA.     

Observation of specific optical phonon modes dominating Li ion diffusion in γ-LiAlO2 ceramic

In γ-LiAlO₂ ceramic, Li ion diffusion plays a key role both in tritium release as a solid tritium breeder material in nuclear fusion and in phase stability as a matrix material in molten carbonate fuel cells (MCFC). Yet fundamental understanding of the diffusive process in γ-LiAlO₂ is still missing, especially considering the interaction of the lattice dynamics and Li ion motion. Herein, we demonstrated that two specific optical phonon modes are coupled with Li ion diffusion in γ-LiAlO₂, by investigating the temperature-dependent lattice dynamics via Raman scattering experiments, as well as first-principles calculations and neutron diffraction experiments (ND). The high-temperature ND experiments showed that γ-LiAlO₂ undergoes partial Li ion disordering in Li sublattice at high temperature. Notably, the lattice dynamics studies demonstrated that B₁ mode at 226 cm^?1 and A₁ mode at 263 cm^?1 are responsible for Li ion motion through the oscillation of Li–O in <010> and <001> direction, respectively. Our results further suggest that the behavior of tritium release and phase stability in γ-LiAlO₂ at elevated temperature may be controlled by tuning the two specific phonon modes through photon/electron-phonon coupling.     

Bacterial 2′-Deoxyguanosine Riboswitch Classes as Potential Targets for Antibiotics: A Structure and Dynamics Study

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.     

Molecular Properties of Human Guanylate Cyclase-Activating Protein 3 (GCAP3) and Its Possible Association with Retinitis Pigmentosa

The cone-specific guanylate cyclase-activating protein 3 (GCAP3), encoded by the GUCA1C gene, has been shown to regulate the enzymatic activity of membrane-bound guanylate cyclases (GCs) in bovine and teleost fish photoreceptors, to an extent comparable to that of the paralog protein GCAP1. To date, the molecular mechanisms underlying GCAP3 function remain largely unexplored. In this work, we report a thorough characterization of the biochemical and biophysical properties of human GCAP3, moreover, we identified an isolated case of retinitis pigmentosa, in which a patient carried the c.301G > C mutation in GUCA1C, resulting in the substitution of a highly conserved aspartate residue by a histidine (p.(D101H)). We found that myristoylated GCAP3 can activate GC1 with a similar Ca²⁺-dependent profile, but significantly less efficiently than GCAP1. The non-myristoylated form did not induce appreciable regulation of GC1, nor did the p.D101H variant. GCAP3 forms dimers under physiological conditions, but at odds with its paralogs, it tends to form temperature-dependent aggregates driven by hydrophobic interactions. The peculiar properties of GCAP3 were confirmed by 2 µs molecular dynamics simulations, which for the p.D101H variant highlighted a very high structural flexibility and a clear tendency to lose the binding of a Ca²⁺ ion to EF3. Overall, our data show that GCAP3 has unusual biochemical properties, which make the protein significantly different from GCAP1 and GCAP2. Moreover, the newly identified point mutation resulting in a substantially unfunctional protein could trigger retinitis pigmentosa through a currently unknown mechanism.     

Synthesis and characterization of soluble polymer ligands possessing sulfide side chains

Soluble polymer ligands possessing aryl monosulfide side chains [poly(4-vinylphenylmethyl sulfide) (PVPMS)], alkyl monosulfide side chains [poly(4-vinylbenzylmethyl sulfide) (PVBMS)] and branched tetrasulfide side chains [poly(7-(4′-vinylbenzyloxy)-2,5,9,12-tetrathiatridecane) (PTeSSD)] were prepared by radical polymerization of the corresponding monomers. These polymer ligands had specific binding abilities for soft acids only, such as Ag(I) and Hg(II) ions. The tetrasulfide-type polymer ligand, PTeSSD, bound the metal ion only by the tetrasulfide side chain itself. The monosulfide-type polymer ligands, PVPMS and PVBMS, bound the metal ion by co-operative interaction between their monosulfide side chains. The resulting polymer-Ag(I) complexes were soluble in organic solvents. Interestingly, a polyester cloth coated with the polymer-Ag(I) complex exhibited significant antibacterial activity against Staphylococcus aureus.