Thiamine Pyrophosphate Riboswitch Research Articles

Identification of a GUAAY Pentaloop Sequence Involved in a Novel RNA Loop–Helix Interaction

Large RNAs often utilize GNRA tetraloops as structural elements to stabilize the overall tertiary fold. These tetraloop–receptor (TR) interactions have a conserved geometry in which the tetraloop docks into the receptor at an angle of ~15° from the helix containing the receptor. Here, we show that the conserved GUAAY pentaloop found in domain III of group IIB1 introns participates in a novel class of RNA tertiary interaction with a geometry and mode of binding that are significantly different from that found in GNRA TR interactions. This pentaloop is highly conserved within the IIB1 class and interacts with the minor groove of the catalytic domain V. The base planes of the loop and receptor nucleotides are not coplanar and greatly deviate from standard A-minor motifs. The helical axis of the GUAAY stem loop diverges ~70° from the angle of insertion found in a typical GNRA TR interaction. Therefore, the loop architecture and insertion orientation are distinctive, with in vitro splicing data indicating that a GNRA tetraloop is incompatible at this position. The GUAAY pentaloop–receptor motif is also found in the structure of the eukaryotic thiamine pyrophosphate riboswitch in the context of a hexanucleotide loop sequence. We therefore propose, based on phylogenetic, structural, and biochemical data, that the GUAAY pentaloop–receptor interaction represents a novel structural motif that is present in multiple structured RNAs.

Genome-wide mapping of transcription start sites yields novel insights into the primary transcriptome of Pseudomonas putida.

The environmental bacterium Pseudomonas putida is an organism endowed with a versatile metabolism and stress tolerance traits that are desirable in an efficient production organism. In this work, differential RNA sequencing was used to investigate the primary transcriptome and RNA regulatory elements of P. putida strain KT2440. A total of 7937 putative transcription start sites (TSSs) were identified, where over two-thirds were located either on the opposite strand or internal to annotated genes. For TSSs associated with mRNAs, sequence analysis revealed a clear Shine-Dalgarno sequence but a lack of conserved overrepresented promoter motifs. These TSSs defined approximately 50 leaderless transcripts and an abundance of mRNAs with long leader regions of which 18 contain RNA regulatory elements from the Rfam database. The thiamine pyrophosphate riboswitch upstream of the thiC gene was examined using an in vivo assay with GFP-fusion vectors and shown to function via a translational repression mechanism. Furthermore, 56 novel intergenic small RNAs and 8 putative actuaton transcripts were detected, as well as 8 novel open reading frames (ORFs). This study illustrates how global mapping of TSSs can yield novel insights into the transcriptional features and RNA output of bacterial genomes.

Utilization of bromine azide to access vicinal-azidobromides from arylidene malononitrile

Abstract An efficient and facile approach for tetrachlorosilane as an in situ mediated transformation via a one-pot, synthesis of vicinal bromoazides through the generation of BrN 3 from azidochlorosilane and N -bromosuccinimide in acetonitrile as solvent at ambient temperature is achieved. This catalytic process represents a highly regioselective and high yielding method for the synthesis of 1,2-bromoazides. Thiamine pyrophosphate (TPP) riboswitches regulate essential genes in bacteria by changing conformation upon binding intracellular TPP. Molecular docking studies are conducted to understand the orientation and the interaction of each synthesized molecules with TPP riboswitches.

Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches

ABSTRACTRiboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.

Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer.

The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The 'Force-FRET' approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity.

In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.

Thiamine pyrophosphate riboswitch in some representative plant species: a bioinformatics study.

Metabolites regulate their own production by directly interacting with highly conserved regions of mRNA that are capable of forming discrete tertiary structures. Such regions of mRNA are called riboswitches. The thiamine pyrophosphate (TPP) riboswitch is the most common riboswitch in different organisms. The TPP is an essential coenzyme that is synthesized by the coupling of pyrimidine (hydroxymethyl pyrimidine) and thiazole (hydroxyethyl thiazole). The TPP riboswitch was searched across all possible phyla of plant kingdom by using Arabidopsis thaliana, a model organism in which TPP riboswitch is already found. The aptameric domain of the TPP riboswitch is conserved at the sequence as well as structure levels in all chosen plant species. The sequence of the noncoding RNA that acts as a riboswitch and is folded into an appropriate stem-loop hairpin secondary structure with minimum free energy is predicted by several computational tools. Most of the secondary structures are similar but not the same, because of variation in sequence size. The TPP ligand can bind to the 3' untranslated region of the aptameric sequence, between the loops P2, P4, and P5 and the region between J2/3 and J4/5. The sequence of these loop regions in all predicted tertiary structure of the riboswitch in representative plant species--green algae to flowering plants--is the same, and the residues situated in these junctions are directly involved in binding thymine pyrophosphate and are conserved in all the representative species.

Small molecules that interact with RNA: riboswitch-based gene control and its involvement in metabolic regulation in plants and algae.

Riboswitches are RNA elements that bind small molecules and in turn regulate gene expression. This mechanism allows the cell to sense the intracellular concentration of these small molecules. A particular riboswitch typically regulates its adjacent gene by altering the transcription, the translation or the splicing of this gene. Recently, a riboswitch that binds thiamin pyrophosphate (TPP) was characterized and found to regulate thiamin biosynthesis in plants and algae. Furthermore, it appears that this element is an essential regulator of primary metabolism in plants. Manipulation of endogenous riboswitch activity resulted in metabolic phenotypes that underlined the role of these elements and their ligands in preserving metabolic homeostasis. This situation supports the hypothesis that riboswitches could be remnants of the most ancient metabolic regulators. Here, we review the mode of action of the plant and algal TPP riboswitch and its relevance to the metabolic network. We also discuss the potential engineering of riboswitches as metabolite sensors in plants and platforms for gene control. Whether additional such RNA-based mechanisms exist in plants and in algae is still an open question, yet, the importance of these elements to metabolic regulation is beyond doubt.

Validating Fragment-Based Drug Discovery for Biological RNAs: Lead Fragments Bind and Remodel the TPP Riboswitch Specifically

Thiamine pyrophosphate (TPP) riboswitches regulate essential genes in bacteria by changing conformation upon binding intracellular TPP. Previous studies using fragment-based approaches identified small molecule "fragments" that bind this gene-regulatory mRNA domain. Crystallographic studies now show that, despite having micromolar Kds, four different fragments bind the TPP riboswitch site-specifically, occupying the pocket that recognizes the aminopyrimidine of TPP. Unexpectedly, the unoccupied site that would recognize the pyrophosphate of TPP rearranges into a structure distinct from that of the cognate complex. This idiosyncratic fragment-induced conformation, also characterized by small-angle X-ray scattering and chemical probing, represents a possible mechanism for adventitious ligand discrimination by the riboswitch, and suggests that off-pathway conformations of RNAs can be targeted for drug development. Our structures, together with previous screening studies, demonstrate the feasibility of fragment-based drug discovery against RNA targets.

Simultaneous Folding of Alternative RNA Structures with Mutual Constraints: An Application to Next-Generation Sequencing-based RNA Structure Probing

Recent advances in next-generation sequencing technology have significantly promoted high-throughput experimental probing of RNA secondary structures. The resulting enzymatic or chemical probing information is then incorporated into a minimum free energy folding algorithm to predict more accurate RNA secondary structures. A drawback of this approach is that it does not consider the presence of alternative RNA structures. In addition, the alternative RNA structures may contaminate experimental probing information of each other and direct the minimum free-energy folding to a wrong direction. In this article, we present a combinatorial solution for this problem, where two alternative structures can be folded simultaneously given the experimental probing information regarding the mixture of these two alternative structures. We have tested our algorithm with artificially generated mixture probing data on adenine riboswitch and thiamine pyrophosphate (TPP) riboswitch. The experimental results show that our algorithm can successfully recover the ON and OFF structures of these riboswitches.

Crystallographic analysis of TPP riboswitch binding by small-molecule ligands discovered through fragment-based drug discovery approaches.

Riboswitches are structured mRNA elements that regulate gene expression in response to metabolite or second-messenger binding and are promising targets for drug discovery. Fragment-based drug discovery methods have identified weakly binding small molecule "fragments" that bind a thiamine pyrophosphate (TPP) riboswitch. However, these fragments require substantial chemical elaboration into more potent, drug-like molecules. Structure determination of the fragments bound to the riboswitch is the necessary next step. In this chapter, we describe the methods for co-crystallization and structure determination of fragment-bound TPP riboswitch structures. We focus on considerations for screening crystallization conditions across multiple crystal forms and provide guidance for building the fragment into the refined crystallographic model. These methods are broadly applicable for crystallographic analyses of any small molecules that bind structured RNAs.

Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch

Structural diversification of canonical nucleic acid bases and nucleotide analogues by tautomerism has been proposed to be a powerful on/off switching mechanism allowing regulation of many biological processes mediated by RNA enzymes and aptamers. Despite the suspected biological importance of tautomerism, attempts to observe minor tautomeric forms in nucleic acid or hybrid nucleic acid-ligand complexes have met with challenges due to the lack of sensitive methods. Here, a combination of spectroscopic, biochemical, and computational tools probed tautomerism in the context of an RNA aptamer-ligand complex; studies involved a model ligand, oxythiamine pyrophosphate (OxyTPP), bound to the thiamine pyrophosphate (TPP) riboswitch (an RNA aptamer) as well as its unbound nonphosphorylated form, oxythiamine (OxyT). OxyTPP, similarly to canonical heteroaromatic nucleic acid bases, has a pyrimidine ring that forms hydrogen bonding interactions with the riboswitch. Tautomerism was established using two-dimensional infrared (2D IR) spectroscopy, variable temperature FTIR and NMR spectroscopies, binding isotope effects (BIEs), and computational methods. All three possible tautomers of OxyT, including the minor enol tautomer, were directly identified, and their distributions were quantitated. In the bound form, BIE data suggested that OxyTPP existed as a 4'-keto tautomer that was likely protonated at the N1'-position. These results also provide a mechanistic framework for understanding the activation of riboswitch in response to deamination of the active form of vitamin B1 (or TPP). The combination of methods reported here revealing the fine details of tautomerism can be applied to other systems where the importance of tautomerism is suspected.

Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity

Thiamin (vitamin B1) is an essential micronutrient needed as a cofactor for many central metabolic enzymes. Animals must have thiamin in their diet, whereas bacteria, fungi, and plants can biosynthesize it de novo from the condensation of a thiazole and a pyrimidine moiety. Although the routes to biosynthesize these two heterocycles are not conserved in different organisms, in all cases exogenous thiamin represses expression of one or more of the biosynthetic pathway genes. One important mechanism for this control is via thiamin-pyrophosphate (TPP) riboswitches, regions of the mRNA to which TPP can bind directly, thus facilitating fine-tuning to maintain homeostasis. However, there is little information on how modulation of riboswitches affects thiamin metabolism in vivo. Here we use the green alga, Chlamydomonas reinhardtii, which regulates both thiazole and pyrimidine biosynthesis with riboswitches in the THI4 (Thiamin 4) and THIC (Thiamin C) genes, respectively, to investigate this question. Our study reveals that regulation of thiamin metabolism is not the simple dogma of negative feedback control. Specifically, balancing the provision of both of the heterocycles of TPP appears to be an important requirement. Furthermore, we show that the Chlamydomonas THIC riboswitch is controlled by hydroxymethylpyrimidine pyrophosphate, as well as TPP, but with an identical alternative splicing mechanism. Similarly, the THI4 gene is responsive to thiazole. The study not only provides insight into the plasticity of the TPP riboswitches but also shows that their maintenance is likely to be a consequence of evolutionary need as a function of the organisms' environment and the particular pathway used.

Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution

Thiamine pyrophosphate (TPP)-sensitive mRNA domains are the most prevalent riboswitches known. Despite intensive investigation, the complex ligand recognition and concomitant folding processes in the TPP riboswitch that culminate in the regulation of gene expression remain elusive. Here, we used single-molecule fluorescence resonance energy transfer imaging to probe the folding landscape of the TPP aptamer domain in the absence and presence of magnesium and TPP. To do so, distinct labeling patterns were used to sense the dynamics of the switch helix (P1) and the two sensor arms (P2/P3 and P4/P5) of the aptamer domain. The latter structural elements make interdomain tertiary contacts (L5/P3) that span a region immediately adjacent to the ligand-binding site. In each instance, conformational dynamics of the TPP riboswitch were influenced by ligand binding. The P1 switch helix, formed by the 5' and 3' ends of the aptamer domain, adopts a predominantly folded structure in the presence of Mg(2+) alone. However, even at saturating concentrations of Mg(2+) and TPP, the P1 helix, as well as distal regions surrounding the TPP-binding site, exhibit an unexpected degree of residual dynamics and disperse kinetic behaviors. Such plasticity results in a persistent exchange of the P3/P5 forearms between open and closed configurations that is likely to facilitate entry and exit of the TPP ligand. Correspondingly, we posit that such features of the TPP aptamer domain contribute directly to the mechanism of riboswitch-mediated translational regulation.

Eukaryotic TPP riboswitch regulation of alternative splicing involving long-distance base pairing

Thiamin pyrophosphate (TPP) riboswitches are found in organisms from all three domains of life. Examples in bacteria commonly repress gene expression by terminating transcription or by blocking ribosome binding, whereas most eukaryotic TPP riboswitches are predicted to regulate gene expression by modulating RNA splicing. Given the widespread distribution of eukaryotic TPP riboswitches and the diversity of their locations in precursor messenger RNAs (pre-mRNAs), we sought to examine the mechanism of alternative splicing regulation by a fungal TPP riboswitch from Neurospora crassa, which is mostly located in a large intron separating protein-coding exons. Our data reveal that this riboswitch uses a long-distance (∼530-nt separation) base-pairing interaction to regulate alternative splicing. Specifically, a portion of the TPP-binding aptamer can form a base-paired structure with a conserved sequence element (α) located near a 5′ splice site, which greatly increases use of this 5′ splice site and promotes gene expression. Comparative sequence analyses indicate that many fungal species carry a TPP riboswitch with similar intron architecture, and therefore the homologous genes in these fungi are likely to use the same mechanism. Our findings expand the scope of genetic control mechanisms relying on long-range RNA interactions to include riboswitches.

Orchestration of Thiamin Biosynthesis and Central Metabolism by Combined Action of the Thiamin Pyrophosphate Riboswitch and the Circadian Clock in Arabidopsis

Riboswitches are natural RNA elements that posttranscriptionally regulate gene expression by binding small molecules and thereby autonomously control intracellular levels of these metabolites. Although riboswitch-based mechanisms have been examined extensively, the integration of their activity with global physiology and metabolism has been largely overlooked. Here, we explored the regulation of thiamin biosynthesis and the consequences of thiamin pyrophosphate riboswitch deficiency on metabolism in Arabidopsis thaliana. Our results show that thiamin biosynthesis is largely regulated by the circadian clock via the activity of the THIAMIN C SYNTHASE (THIC) promoter, while the riboswitch located at the 3' untranslated region of this gene controls overall thiamin biosynthesis. Surprisingly, the results also indicate that the rate of thiamin biosynthesis directs the activity of thiamin-requiring enzymes and consecutively determines the rate of carbohydrate oxidation via the tricarboxylic acid cycle and pentose-phosphate pathway. Our model suggests that in Arabidopsis, the THIC promoter and the thiamin-pyrophosphate riboswitch act simultaneously to tightly regulate thiamin biosynthesis in a circadian manner and consequently sense and control vital points of core cellular metabolism.

Observation of Intrinsic Conformational Dynamics of TPP Riboswitch and its Structural Transition Induced by TPP Binding

A riboswitch is a non-coding region of an mRNA which recognizes a specific metabolite and regulates the expression of the mRNA itself. They are composed of two domains: an aptamer domain which recognizes a metabolite, and downstream expression platform. It is believed that metabolite binding to the aptamer domain induces a structural change of the riboswitch, signaling “on” or “off” for the transcription or translation of the mRNA.Recent publications of a number of high-resolution structures of ligand-bound aptamer domains (halo-form) greatly contributed to our understanding of how the riboswitches interact with their natural ligands at the atomic-level. However, little is known about the nature of the unbound form of riboswitches (apo-form), and how conformational changes of riboswitches is induced by binding of a ligand to the aptamer domain.We used fluorescence resonance energy transfer (FRET) to study conformational dynamics of the Escherichia coli thiM TPP (thiamine pyrophosphate) riboswitch. We found that even though the apo-aptamer dynamically samples a closed conformation resembling the halo-aptamer, both the open form and the closed form recognize the ligand with equal preference. And the final structural transition of the riboswitch induced by the ligand is also observed. Both indicate that the ligand is recognized via the induced-fit mechanism. Furthermore, the role of Mg ions on the ligand recognition mechanism will be also discussed.

Thermodynamic Examination of Bulge Sequence, Ion Binding, and Helical Context of 2X2 and 2X1 Bulges in RNA

Bulges are non‐helical regions in RNA that provide sites for protein and ligand interactions. The interactions in and around the bulge region are non‐canonical and often not well understood. In crystal structures, bulges and non‐canonical base pairs are often the sites for interactions with metal ions. The pyrophosphate sensor helix of the thiamine pyrophosphate (TPP) riboswitch contains a 2×2 bulge that binds the pyrophosphate group along with Mg2+ ions. The 1×2 internal stem loop of the U6 spliceosomal RNA changes conformation upon the formation of A+•C base pair and Mg2+ binding. In this project, we are investigating the non‐canonical base pairs in the bulge regions, the helical context in which these are found, and the stabilization of these RNA in the presence of Mg2+ ions. Wild type and modified RNA constructs were designed to study RNA stability using thermal denaturation experiments in 1 M KCl and in varying concentrations of Mg2+, in pH 5.5 and 7.5. In 1 M KCl, the wild type U6 2X1 and TPP 2X2 constructs showed a greater stability at pH 5.5 over pH 7.5 likely due to the formation of A+•C base pair. All TPP 2X2 constructs, with one exception, are more stable in 9.5 mM Mg2+ over 1 M KCl. Comparative thermodynamic data will be presented on various constructs to better elucidate the differences and similarities of 2X2 and 1×2 bulge regions. This work was funded by NSF Grant MCB‐0950582 to NG.

Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer

Riboswitches are motifs in the untranslated regions (UTRs) of RNA transcripts that sense metabolite levels and modulate the expression of the corresponding genes for metabolite import, export, synthesis, or degradation. All riboswitches contain an aptamer: an RNA structure that, upon binding ligand, folds to expose or sequester regulatory elements in the adjacent sequence through alternative nucleotide pairing. The coupling between ligand binding and aptamer folding is central to the regulatory mechanisms of thiamine pyrophosphate (TPP) riboswitches and has not been fully characterized. Here, we show that TPP aptamer folding can be decomposed into ligand-independent and -dependent steps that correspond to the formation of secondary and tertiary structures, respectively. We reconstructed the full energy landscape for folding of the wild-type (WT) aptamer and measured perturbations of this landscape arising from mutations or ligand binding. We show that TPP binding proceeds in two steps, from a weakly to a strongly bound state. Our data imply a hierarchical folding sequence, and provide a framework for understanding molecular mechanism throughout the TPP riboswitch family.

Rational Design of a New IMP Aptamer Based on a TPP Riboswitch and a Hypoxanthine Aptamer

Abstract We rationally designed a new inosine monophosphate (IMP) aptamer based on structural information from the natural thiamine pyrophosphate (TPP) riboswitch and an in vitro-selected hypoxanthine aptamer. The value of the binding affinity (Ka) for the newly designed IMP aptamer for IMP was (2.6 ± 3.2) × 107 M−1, which was 10-times larger than that for TPP. These results demonstrated that structure-based design of aptamers can be a useful scheme to develop artificial aptamers.