Impacts of Fluorescent Base Analogue Substitution on the Folding of a Riboswitch

Riboswitches are gene-regulating mRNA segments most commonly found in bacteria. A riboswitch contains an aptamer domain that binds to a ligand, causing a conformational change in a downstream expression platform. The aptamer domain of the Class I preQ1 riboswitch from Bacillus subtilis, which consists of a stem-loop structure and an adenine-rich single-stranded tail (L3), re-folds into a pseudoknot structure upon binding of its ligand, preQ1. To study the role of L3 in ligand recognition, we inserted 2-aminopurine (2-AP), a fluorescent base analogue of adenine (A), into the riboswitch at six different positions within L3. 2-AP differs from A in the relocation of its amino group from C6 to C2, allowing us to directly probe the significance of this specific functional group. We used circular dichroism spectroscopy and thermal denaturation experiments to study the structure and stability, respectively, of the riboswitch in the absence and presence of preQ1. At all labeling positions tested, 2-AP substitution inhibited the ability of preQ1 to stabilize the pseudoknot structure, with its location impacting the severity of the effect. Structural studies of the riboswitch suggest that at the most detrimental labeling sites, 2-AP substitution disrupts non-canonical base pairs. Our results show that these base pairs and tertiary interactions involving other residues in L3 play a critical role in ligand recognition by the preQ1 riboswitch, even at positions that are distal to the ligand binding pocket. They also highlight the importance of accounting for perturbations that fluorescent analogues like 2-AP may exert on the system being studied.

Ensemble Switching Unveils a Kinetic Rheostat Mechanism of the Eukaryotic Thiamine Pyrophosphate Riboswitch

ABSTRACTThiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on theArabidopsis thalianaTPP riboswitch suggests a multi-step TPP binding process involving multiple riboswitch conformational ensembles and that Mg2+dependence underlies the mechanism of TPP recognition and subsequent transition to the translation-inhibiting state of the switching sequence followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg2+in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain associated with TPP and Mg2+ligand binding occurred between at least five different ensembles on timescales ranging from μs to ms. These dynamics are at least an order of magnitude faster than folding and unfolding kinetics associated with translation-state switching in the switching sequence. Moreover, we propose that two pathways exist for ligand recognition. Together, our results suggest a dynamic ensemble switching of the aptamer domain that may lead to the translation-inhibiting state of the riboswitch. Additionally, our results suggest that multiple configurations could enable inhibitory tuning manifested through ligand-dependent changes via ensemble switching and kinetic rheostat-like behavior of theArabidopsis thalianaTPP riboswitch.

Requirements for efficient ligand-gated co-transcriptional switching in designed variants of the B. subtilis pbuE adenine-responsive riboswitch in E. coli

Riboswitches, generally located in the 5’-leader of bacterial mRNAs, direct expression via a small molecule-dependent structural switch that informs the transcriptional or translational machinery. While the structure and function of riboswitch effector-binding (aptamer) domains have been intensely studied, only recently have the requirements for efficient linkage between small molecule binding and the structural switch in the cellular and co-transcriptional context begun to be actively explored. To address this aspect of riboswitch function, we have performed a structure-guided mutagenic analysis of the B. subtilis pbuE adenine-responsive riboswitch, one of the simplest riboswitches that employs a strand displacement switching mechanism to regulate transcription. Using a cell-based fluorescent protein reporter assay to assess ligand-dependent regulatory activity in E. coli, these studies revealed previously unrecognized features of the riboswitch. Within the aptamer domain, local and long-range conformational dynamics influenced by sequences within helices have a significant effect upon efficient regulatory switching. Sequence features of the expression platform including the pre-aptamer leader sequence, a toehold helix and an RNA polymerase pause site all serve to promote strong ligand-dependent regulation. By optimizing these features, we were able to improve the performance of the B. subtilis pbuE riboswitch in E. coli from 5.6-fold induction of reporter gene expression by the wild type riboswitch to over 120-fold in the top performing designed variant. Together, these data point to sequence and structural features distributed throughout the riboswitch required to strike a balance between rates of ligand binding, transcription and secondary structural switching via a strand exchange mechanism and yield new insights into the design of artificial riboswitches.

Thiamine pyrophosphate riboswitch regulation: a new possible mechanism involved in the action of nalidixic acid

ObjectivesThe development of novel antibiotic compounds requires riboswitches; in fact, riboswitches are RNA elements present in the 5′ untranslated region of bacterial mRNA and have a metabolite-binding aptamer domain and an expression platform regulating the expression of vital genes. In the present research, one riboswitch, namely thi-box riboswitch with distinct regulatory mechanisms, was studied. It recognizes Thiamine Pyrophosphates (TPP) regulating TPP-biosynthesis genes in Escherichiacoli.MethodsFirst, the compounds similar to riboswitch ligands were studied, and their binding with the riboswitch and nucleosides was investigated by molecular docking. Then, compounds containing high binding energy were chosen, and their minimum inhibitory concentration in E. coli was determined by the MIC test. Finally, the binding of compounds to nucleotides and RNA was investigated by measuring the absorbance spectrum through NanoDrop and circular dichroism (CD).ResultsIn the thi-box riboswitch, nalidixic acid was found to have the best binding energy (−5.31 kJ/mol), and it inhibited E. coli growth at the minimum inhibitory concentration of 125 μg/mL, and it could bind to ribonucleosides and RNA in vitro.ConclusionsOne possible mechanism involved in the action of nalidixic acid in inhibiting the E. coli growth is to influence thi-box riboswitch.

The conformational landscape of transcription intermediates involved in the regulation of the ZMP-sensing riboswitch from Thermosinus carboxydivorans

Recently, prokaryotic riboswitches have been identified that regulate transcription in response to change of the concentration of secondary messengers. The ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR))-sensing riboswitch from Thermosinus carboxydivorans is a transcriptional ON-switch that is involved in purine and carbon-1 metabolic cycles. Its aptamer domain includes the pfl motif, which features a pseudoknot, impeding rho-independent terminator formation upon stabilization by ZMP interaction. We herein investigate the conformational landscape of transcriptional intermediates including the expression platform of this riboswitch and characterize the formation and unfolding of the important pseudoknot structure in the context of increasing length of RNA transcripts. NMR spectroscopic data show that even surprisingly short pre-terminator stems are able to disrupt ligand binding and thus metabolite sensing. We further show that the pseudoknot structure, a prerequisite for ligand binding, is preformed in transcription intermediates up to a certain length. Our results describe the conformational changes of 13 transcription intermediates of increasing length to delineate the change in structure as mRNA is elongated during transcription. We thus determine the length of the key transcription intermediate to which addition of a single nucleotide leads to a drastic drop in ZMP affinity.

Structural Ensembles of Ribonucleic Acids From Solvent Accessibility Data: Application to the S-Adenosylmethionine (SAM)-Responsive Riboswitch

ABSTRACTRiboswitches are regulatory ribonucleic acid (RNA) elements that act as ligand-dependent conformational switches. In the apo form, the aptamer domain, the region of a riboswitch that binds to its cognate ligand, is dynamic, thus requiring an ensemble-representation of its structure. Analysis of such ensembles can provide molecular insights into the sensing mechanism and capabilities of riboswitches. Here, as a proof-of-concept, we constructed a pair of atomistic ensembles of the well-studied S-adenosylmethionine (SAM)-responsive riboswitch in the absence (-SAM) and presence (+SAM) of SAM. To achieve this, we first generated a large conformational pool and then reweighted conformers in the pool using solvent accessible surface area (SASA) data derived from recently reported light-activated structural examination of RNA (LASER) reactivities, measured in the −SAM and +SAM states of the riboswitch. The differences in the resulting −SAM and +SAM ensembles are consistent with a SAM-dependent reshaping of the free landscape of the riboswitch. Interestingly, within the −SAM ensemble, we identified a conformer that harbors a hidden binding pocket, which was discovered using ensemble docking. The method we have applied to the SAM riboswitch is general, and could, therefore, be used to construct atomistic ensembles for other riboswitches, and more broadly, other classes of structured RNAs.

A 300-fold enhancement of imino nucleic acid resonances by hyperpolarized water provides a new window for probing RNA refolding by 1D and 2D NMR

NMR sensitivity-enhancement methods involving hyperpolarized water could be of importance for solution-state biophysical investigations. Hyperpolarized water (HyperW) can enhance the 1H NMR signals of exchangeable sites by orders of magnitude over their thermal counterparts, while providing insight into chemical exchange and solvent accessibility at a site-resolved level. As HyperW’s enhancements are achieved by exploiting fast solvent exchanges associated with minimal interscan delays, possibilities for the rapid monitoring of chemical reactions and biomolecular (re)folding are opened. HyperW NMR can also accommodate heteronuclear transfers, facilitating the rapid acquisition of 2-dimensional (2D) 15N-1H NMR correlations, and thereby combining an enhanced spectral resolution with speed and sensitivity. This work demonstrates how these qualities can come together for the study of nucleic acids. HyperW injections were used to target the guanine-sensing riboswitch aptamer domain (GSRapt) of the xpt-pbuX operon in Bacillus subtilis. Unlike what had been observed in proteins, where residues benefited of HyperW NMR only if/when sufficiently exposed to water, these enhancements applied to every imino resonance throughout the RNA. The >300-fold enhancements observed in the resulting 1H NMR spectra allowed us to monitor in real time the changes that GSRapt undergoes upon binding hypoxanthine, a high-affinity interaction leading to conformational refolding on a ∼1-s timescale at 36 °C. Structural responses could be identified for several nucleotides by 1-dimensional (1D) imino 1H NMR as well as by 2D HyperW NMR spectra acquired upon simultaneous injection of hyperpolarized water and hypoxanthine. The folding landscape revealed by this HyperW strategy for GSRapt, is briefly discussed.

Structural basis for 2′-deoxyguanosine recognition by the 2′-dG-II class of riboswitches

A recent bioinformatic analysis of well-characterized classes of riboswitches uncovered subgroups unable to bind to the regulatory molecule of the parental class. Within the guanine/adenine class, seven groups of RNAs were identified that deviate from the consensus sequence at one or more of three positions directly involved purine nucleobase recognition, one of which was validated as a second class of 2′-deoxyguanosine riboswitch (called 2′-dG-II). To understand how 2′-dG-II riboswitches recognize their cognate ligand and how they differ from a previously identified class of 2′-deoxyguanosine binding riboswitches, we have solved the crystal structure of a 2′-dG-II aptamer domain bound to 2′-deoxyguanosine. This structure reveals a global architecture similar to other members of the purine riboswitch family, but contains key differences within the ligand binding core. Defining the 2′-dG-II riboswitches is a two-nucleotide insertion in the three-way junction that promotes novel base-base interactions. Unlike 2′-dG-I riboswitches, the 2′-dG-II class only requires local changes to the ligand binding pocket of the guanine/adenine class to achieve a change in ligand preference. Notably, members of the 2′-dG-II family have variable ability to discriminate between 2′-deoxyguanosine and riboguanosine, suggesting that a subset of 2′-dG-II riboswitches may bind either molecule to regulate gene expression.