The Impact of Second-Shell Nucleotides on Ligand Specificity in Cyclic Dinucleotide Riboswitches.

Ligand specificity is an essential requirement for all riboswitches. Some variant riboswitches utilize a common structural motif, yet through subtle sequence differences, they are able to selectively respond to different small molecule ligands and regulate downstream gene expression. These variants discriminate between structurally and chemically similar ligands. Crystal structures provide insight into how specificity is achieved. However, ligand specificity cannot always be explained solely by nucleotides in direct contact with the ligand. The cyclic dinucleotide variant family contains two classes, cyclic-di-GMP and cyclic-AMP-GMP riboswitches, that were distinguished based on the identity of a single nucleotide in contact with the ligand. Here we report a variant riboswitch with a mutation at a second ligand-contacting position that is promiscuous for both cyclic-di-GMP and cyclic-AMP-GMP despite a predicted preference for cyclic-AMP-GMP. A high-throughput mutational analysis, SMARTT, was used to quantitatively assess thousands of sites in the first- and second-shells of ligand contact for impacts on ligand specificity and promiscuity. In addition to nucleotides in direct ligand contact, nucleotides more distal from the binding site, within the J1/2 linker and the terminator helix, were identified that impact ligand specificity. These findings provide an example of how nucleotides outside the ligand binding pocket influence the riboswitch specificity. Moreover, these distal nucleotides could be used to predict promiscuous sequences.

Fluoride transport in Arabidopsis thaliana plants is impaired in Fluoride EXporter (FEX) mutants.

Fluoride is an environmental toxin prevalent in water, soil, and air. A fluoride transporter called Fluoride EXporter (FEX) has been discovered across all domains of life, including bacteria, single cell eukaryotes, and all plants, that is required for fluoride tolerance. How FEX functions to protect multicellular plants is unknown. In order to distinguish between different models, the dynamic movement of fluoride in wildtype (WT) and fex mutant plants was monitored using [18F]fluoride with positron emission tomography. Significant differences were observed in the washout behavior following initial fluoride uptake between plants with and without a functioning FEX. [18F]Fluoride traveled quickly up the floral stem and into terminal tissues in WT plants. In contrast, the fluoride did not move out of the lower regions of the stem in mutant plants resulting in clearance rates near zero. The roots were not the primary locus of FEX action, nor did FEX direct fluoride to a specific tissue. Fluoride efflux by WT plants was saturated at high fluoride concentrations resulting in a pattern like the fex mutant. The kinetics of fluoride movement suggested that FEX mediates a fluoride transport mechanism throughout the plant where each individual cell benefits from FEX expression.

Translation regulation by a guanidine-II riboswitch is highly tunable in sensitivity, dynamic range, and apparent cooperativity.

Riboswitches function as important translational regulators in bacteria. Comprehensive mutational analysis of transcriptional riboswitches has been used to probe the energetic intricacies of interplay between the aptamer and expression platform, but translational riboswitches have been inaccessible to massively parallel techniques. The guanidine-II (gdm-II) riboswitch is an exclusively translational class. We have integrated RelE cleavage with next-generation sequencing to quantify ligand-dependent changes in translation initiation for all single and double mutations of the Pseudomonas aeruginosa gdm-II riboswitch, a total of more than 23,000 variants. This extensive mutational analysis is consistent with the prominent features of the bioinformatic consensus. These data indicate, unexpectedly, that direct sequestration of the Shine-Dalgarno sequence is dispensable for riboswitch function. Additionally, this comprehensive data set reveals important positions not identified in previous computational and crystallographic studies. Mutations in the variable linker region stabilize alternate conformations. The double mutant data reveal the functional importance of the previously modeled P0b helix formed by the 5' and 3' tails that serves as the basis for translational control. Additional mutations to GU wobble base pairs in both P1 and P2 reveal how the apparent cooperativity of the system involves an intricate network of communication between the two binding sites. This comprehensive examination of a translational riboswitch's expression platform illuminates how the riboswitch is precisely tuned and tunable with regard to ligand sensitivity, the amplitude of expression between ON and OFF states, and the cooperativity of ligand binding.

Cells Adapt to Resist Fluoride through Metabolic Deactivation and Intracellular Acidification.

Fluoride is highly abundant in the environment. Many organisms have adapted specific defense mechanisms against high concentrations of fluoride, including the expression of proteins capable of removing fluoride from cells. However, these fluoride transporters have not been identified in all organisms, and even organisms that express fluoride transporters vary in tolerance capabilities across species, individuals, and even tissue types. This suggests that alternative factors influence fluoride tolerance. We screened for adaptation against fluoride toxicity through an unbiased mutagenesis assay conducted on Saccharomyces cerevisiae lacking the fluoride exporter FEX, the primary mechanism of fluoride resistance. Over 80 independent fluoride-hardened strains were generated, with anywhere from 100- to 1200-fold increased fluoride tolerance compared to the original strain. The whole genome of each mutant strain was sequenced and compared to the wild type. The fluoride-hardened strains utilized a combination of phenotypes that individually conferred fluoride tolerance. These included intracellular acidification, cellular dormancy, nutrient storage, and a communal behavior reminiscent of flocculation. Of particular importance to fluoride resistance was intracellular acidification, which served to reverse the accumulation of fluoride and lead to its excretion from the cell as HF without the activity of a fluoride-specific protein transporter. This transport mechanism was also observed in wild-type yeast through a manual mutation to lower their cytoplasmic pH. The results demonstrate that the yeast developed a protein-free adaptation for removing an intracellular toxicant.

Efficient quantitative monitoring of translational initiation by RelE cleavage.

The sequences of the 5' untranslated regions (5'-UTRs) of mRNA alter gene expression across domains of life. Transcriptional modulators can be easily assayed through transcription termination, but translational regulators often require indirect, laborious methods. We have leveraged RelE's ribosome-dependent endonuclease activity to develop a quantitative assay to monitor translation initiation of cis-regulatory mRNAs. RelE cleavage accurately reports ligand-dependent changes in ribosome association for two translational riboswitches and provides quantitative information about each switch's sensitivity and range of response. RelE accurately reads out sequence-driven changes in riboswitch specificity and function and is quantitatively dependent upon ligand concentration. RelE cleavage similarly captures differences in translation initiation between yeast 5'-UTR isoforms. RelE cleavage can thus reveal a plethora of information about translation initiation in different domains of life.

The fluoride transporter FLUORIDE EXPORTER (FEX) is the major mechanism of tolerance to fluoride toxicity in plants.

Fluoride is everywhere in the environment, yet it is toxic to living things. How biological organisms detoxify fluoride has been unknown until recently. Fluoride-specific ion transporters in both prokaryotes (Fluoride channel; Fluc) and fungi (Fluoride Exporter; FEX) efficiently export fluoride to the extracellular environment. FEX homologues have been identified throughout the plant kingdom. Understanding the function of FEX in a multicellular organism will reveal valuable knowledge about reducing toxic effects caused by fluoride. Here we demonstrate the conserved role of plant FEX (FLUORIDE EXPORTER) in conferring fluoride tolerance. Plant FEX facilitates the efflux of toxic fluoride ions from yeast cells and is required for fluoride tolerance in plants. A CRISPR/Cas9-generated mutation in Arabidopsis thaliana FEX renders the plant vulnerable to low concentrations (100 µM) of fluoride at every stage of development. Pollen is particularly affected, failing to develop even at extremely low levels of fluoride in the growth medium. The action of the FEX membrane transport protein is the major fluoride defense mechanism in plants.

The fluoride transporter FLUORIDE EXPORTER (FEX) is the major mechanism of tolerance to fluoride toxicity in plants1.

Fluoride is everywhere in the environment, yet it is toxic to living things. How biological organisms detoxify fluoride has been unknown until recently. Fluoride-specific ion transporters in both prokaryotes (Fluoride channel; Fluc) and fungi (Fluoride Exporter; FEX) efficiently export fluoride to the extracellular environment. FEX homologs have been identified throughout the plant kingdom. Understanding the function of FEX in a multicellular organism will reveal valuable knowledge about reducing toxic effects caused by fluoride. Here, we demonstrate the conserved role of plant FEX (FLUORIDE EXPORTER) in conferring fluoride tolerance. Plant FEX facilitates the efflux of toxic fluoride ions from yeast cells and is required for fluoride tolerance in plants. A CRISPR/Cas9-generated mutation in Arabidopsis thaliana FEX renders the plant vulnerable to low concentrations (100-µM) of fluoride at every stage of development. Pollen is particularly affected, failing to develop even at extremely low levels of fluoride in the growth medium. The action of the FEX membrane transport protein is the major fluoride defense mechanism in plants.

How alginate properties influence in situ internal gelation in crosslinked alginate microcapsules (CLAMs) formed by spray drying.

Alginates gel rapidly under ambient conditions and have widely documented potential to form protective matrices for sensitive bioactive cargo. Most commonly, alginate gelation occurs via calcium mediated electrostatic crosslinks between the linear polyuronic acid polymers. A recent breakthrough to form crosslinked alginate microcapsules (CLAMs) by in situ gelation during spray drying ("CLAMs process") has demonstrated applications in protection and controlled delivery of bioactives in food, cosmetics, and agriculture. The extent of crosslinking of alginates in CLAMs impacts the effectiveness of its barrier properties. For example, higher crosslinking extents can improve oxidative stability and limit diffusion of the encapsulated cargo. Crosslinking in CLAMs can be controlled by varying the calcium to alginate ratio; however, the choice of alginates used in the process also influences the ultimate extent of crosslinking. To understand how to select alginates to target crosslinking in CLAMs, we examined the roles of alginate molecular properties. A surprise finding was the formation of alginic acid gelling in the CLAMs that is a consequence of simultaneous and rapid pH reduction and moisture removal that occurs during spray drying. Thus, spray dried CLAMs gelation is due to calcium crosslinking and alginic acid formation, and unlike external gelation methods, is insensitive to the molecular composition of the alginates. The 'extent of gelation' of spray dried CLAMs is influenced by the molecular weights of the alginates at saturating calcium concentrations. Alginate viscosity correlates with molecular weight; thus, viscosity is a convenient criterion for selecting commercial alginates to target gelation extent in CLAMs.

Genome-Wide Identification of Genes Involved in General Acid Stress and Fluoride Toxicity in Saccharomyces cerevisiae.

Hydrofluoric acid elicits cell cycle arrest through a mechanism that has long been presumed to be linked with the high affinity of fluoride to metals. However, we have recently found that the acid stress from fluoride exposure is sufficient to elicit many of the hallmark phenotypes of fluoride toxicity. Here we report the systematic screening of genes involved in fluoride resistance and general acid resistance using a genome deletion library in Saccharomyces cerevisiae. We compare these to a variety of acids - 2,4-dinitrophenol, FCCP, hydrochloric acid, and sulfuric acid - none of which has a high metal affinity. Pathways involved in endocytosis, vesicle trafficking, pH maintenance, and vacuolar function are of particular importance to fluoride tolerance. The majority of genes conferring resistance to fluoride stress also enhanced resistance to general acid toxicity. Genes whose expression regulate Golgi-mediated vesicle transport were specific to fluoride resistance, and may be linked with fluoride-metal interactions. These results support the notion that acidity is an important and underappreciated principle underlying the mechanisms of fluoride toxicity.

A DNA Repair Inhibitor Isolated from an Ecuadorian Fungal Endophyte Exhibits Synthetic Lethality in PTEN-Deficient Glioblastoma.

Disruption of the tumor suppressor PTEN, either at the protein or genomic level, plays an important role in human cancer development. The high frequency of PTEN deficiency reported across several cancer subtypes positions therapeutic approaches that exploit PTEN loss-of-function with the ability to significantly impact the treatment strategies of a large patient population. Here, we report that an endophytic fungus isolated from a medicinal plant produces an inhibitor of DNA double-strand-break repair. Furthermore, the novel alkaloid product, which we have named irrepairzepine (1), demonstrated synthetic lethal targeting in PTEN-deficient glioblastoma cells. Our results uncover a new therapeutic lead for PTEN-deficient cancers and an important molecular tool toward enhancing the efficacy of current cancer treatments.

The Positively Charged Active Site of the Bacterial Toxin RelE Causes a Large Shift in the General Base pKa.

The bacterial toxin RelE cleaves mRNA in the ribosomal A site. Although it shares a global fold with other microbial RNases, the active site contains several positively charged residues instead of histidines and glutamates that are typical of ribonucleases. The pH dependences of wild-type and mutant RelE indicate it uses general acid-base catalysis, but either the general acid (proposed to be R81) or the general base must have a substantially downshifted pKa. However, which group is shifted cannot be determined using available structural and biochemical data. Here, we use a phosphorothiolate at the scissile phosphate to remove the need for a general acid. We show this modification rescues nearly all of the defect of the R81A mutation, supporting R81 as the general acid. We also find that the observed pKa of the general base is dependent on the charge of the side chain at position 81. This indicates that positive charge in the active site contributes to a general base pKa downshifted by more than 5 units. Although this modestly reduces the effectiveness of general acid-base catalysis, it is strongly supplemented by the role of the positive charge in stabilizing the transition state for cleavage. Furthermore, we show that the ribosome is required for cleavage but not binding of mRNA by RelE. Ribosome functional groups do not directly contact the scissile phosphate, indicating that positioning and charge interactions dominate RelE catalysis. The unusual RelE active site catalyzes phosphoryl transfer at a rate comparable to those of similar enzymes, but in a ribosome-dependent fashion.

Principles of fluoride toxicity and the cellular response: a review.

Fluoride is ubiquitously present throughout the world. It is released from minerals, magmatic gas, and industrial processing, and travels in the atmosphere and water. Exposure to low concentrations of fluoride increases overall oral health. Consequently, many countries add fluoride to their public water supply at 0.7-1.5 ppm. Exposure to high concentrations of fluoride, such as in a laboratory setting often exceeding 100 ppm, results in a wide array of toxicity phenotypes. This includes oxidative stress, organelle damage, and apoptosis in single cells, and skeletal and soft tissue damage in multicellular organisms. The mechanism of fluoride toxicity can be broadly attributed to four mechanisms: inhibition of proteins, organelle disruption, altered pH, and electrolyte imbalance. Recently, there has been renewed concern in the public sector as to whether fluoride is safe at the current exposure levels. In this review, we will focus on the impact of fluoride at the chemical, cellular, and multisystem level, as well as how organisms defend against fluoride. We also address public concerns about fluoride toxicity, including whether fluoride has a significant effect on neurodegeneration, diabetes, and the endocrine system.

A Modular RNA Domain That Confers Differential Ligand Specificity.

The modularity of protein domains is well-known, but the existence of independent domains that confer function in RNA is less established. Recently, a family of RNA aptamers termed ykkC was discovered; they bind at least four ligands of very different chemical composition, including guanidine, phosphoribosyl pyrophosphate (PRPP), and guanosine tetraphosphate (ppGpp) (graphical abstract). Structures of these aptamers revealed an architecture characterized by two coaxial helical stacks. The first helix appears to be a generic scaffold, while the second helix forms the most contacts to the ligands. To determine if these two regions within the aptamer are modular units for ligand recognition, we swapped the ligand-binding coaxial stacks of a guanidine aptamer and a PRPP aptamer. This operation, in combination with a single mutation in the scaffold domain, achieved full switching of ligand specificity. This finding suggests that the ligand-binding helix largely dictates the ligand specificity of ykkC RNAs and that the scaffold coaxial stack is generally compatible with various ykkC ligand-binding modules. This work presents an example of RNA domain modularity comparable to that of a ligand-binding protein, showcasing the versatility of RNA as an entity capable of molecular evolution through adaptation of existing motifs.

The asymmetry and cooperativity of tandem glycine riboswitch aptamers.

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K1/2, while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae, which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches.

Nitrate and Phosphate Transporters Rescue Fluoride Toxicity in Yeast.

Organisms are exposed to fluoride in the air, water, and soil. Yeast and other microbes utilize fluoride channels as a method to prevent intracellular fluoride accumulation and mediate fluoride toxicity. Consequently, deletion of fluoride exporter genes (FEX) in S. cerevisiae resulted in over 1000-fold increased fluoride sensitivity. We used this FEX knockout strain to identify genes, that when overexpressed, are able to partially relieve the toxicity of fluoride exposure. Overexpression of five genes, SSU1, YHB1, IPP1, PHO87, and PHO90, increase fluoride tolerance by 2- to 10-fold. Overexpression of these genes did not provide improved fluoride resistance in wild-type yeast, suggesting that the mechanism is specific to low fluoride toxicity in yeast. Ssu1p and Yhb1p both function in nitrosative stress response, which is induced upon fluoride exposure along with metal influx. Ipp1p, Pho87p, and Pho90p increase intracellular orthophosphate. Consistent with this observation, fluoride toxicity is also partially mitigated by the addition of high levels of phosphate to the growth media. Fluoride inhibits phosphate import upon stress induction and causes nutrient starvation and organelle disruption, as supported by gene induction monitored through RNA-Seq. The combination of observations suggests that transmembrane nutrient transporters are among the most sensitized proteins during fluoride-instigated stress.

Gene regulation by a glycine riboswitch singlet uses a finely tuned energetic landscape for helical switching.

Riboswitches contain structured aptamer domains that, upon ligand binding, facilitate helical switching in their downstream expression platforms to alter gene expression. To fully dissect how riboswitches function requires a better understanding of the energetic landscape for helical switching. Here, we report a sequencing-based high-throughput assay for monitoring in vitro transcription termination and use it to simultaneously characterize the functional effects of all 522 single point mutants of a glycine riboswitch type-1 singlet. Mutations throughout the riboswitch cause ligand-dependent defects, but only mutations within the terminator hairpin alter readthrough efficiencies in the absence of ligand. A comprehensive analysis of the expression platform reveals that ligand binding stabilizes the antiterminator by just 2-3 kcal/mol, indicating that the competing expression platform helices must be extremely close in energy to elicit a significant ligand-dependent response. These results demonstrate that gene regulation by this riboswitch is highly constrained by the energetics of ligand binding and conformational switching. These findings exemplify the energetic parameters of RNA conformational rearrangements driven by binding events.

Industrially-Scalable Microencapsulation of Plant Beneficial Bacteria in Dry Cross-Linked Alginate Matrix.

Microencapsulation of plant-beneficial bacteria, such as pink pigmented facultative methylotrophs (PPFM), may greatly extend the shelf life of these Gram-negative microorganisms and facilitate their application to crops for sustainable agriculture. A species of PPFM designated Methylobacterium radiotolerans was microencapsulated in cross-linked alginate microcapsules (CLAMs) prepared by an innovative and industrially scalable process that achieves polymer cross-linking during spray-drying. PPFM survived the spray-drying microencapsulation process with no significant loss in viable population, and the initial population of PPFM in CLAMs exceeded 1010 CFU/g powder. The PPFM population in CLAMs gradually declined by 4 to 5 log CFU/g over one year of storage. The extent of alginate cross-linking, modulated by adjusting the calcium phosphate content in the spray-dryer feed, did not influence cell viability after spray-drying, viability over storage, or dry particle size. However, particle size measurements and light microscopy of aqueous CLAMs suggest that enhanced crosslinking may limit the release of encapsulated bacteria. This work demonstrates an industrially scalable method for producing alginate-based inoculants that may be suitable for on-seed or foliar spray applications.

Structures of two aptamers with differing ligand specificity reveal ruggedness in the functional landscape of RNA.

Two classes of riboswitches related to the ykkC guanidine-I riboswitch bind phosphoribosyl pyrophosphate (PRPP) and guanosine tetraphosphate (ppGpp). Here we report the co-crystal structure of the PRPP aptamer and its ligand. We also report the structure of the G96A point mutant that prefers ppGpp over PRPP with a dramatic 40,000-fold switch in specificity. The ends of the aptamer form a helix that is not present in the guanidine aptamer and is involved in the expression platform. In the mutant, the base of ppGpp replaces G96 in three-dimensional space. This disrupts the S-turn, which is a primary structural feature of the ykkC RNA motif. These dramatic differences in ligand specificity are achieved with minimal mutations. ykkC aptamers are therefore a prime example of an RNA fold with a rugged fitness landscape. The ease with which the ykkC aptamer acquires new specificity represents a striking case of evolvability in RNA.

Enzymatic synthesis of cyclic dinucleotide analogs by a promiscuous cyclic-AMP-GMP synthetase and analysis of cyclic dinucleotide responsive riboswitches.

Cyclic dinucleotides are second messenger molecules produced by both prokaryotes and eukaryotes in response to external stimuli. In bacteria, these molecules bind to RNA riboswitches and several protein receptors ultimately leading to phenotypic changes such as biofilm formation, ion transport and secretion of virulence factors. Some cyclic dinucleotide analogs bind differentially to biological receptors and can therefore be used to better understand cyclic dinucleotide mechanisms in vitro and in vivo. However, production of some of these analogs involves lengthy, multistep syntheses. Here, we describe a new, simple method for enzymatic synthesis of several 3', 5' linked cyclic dinucleotide analogs of c-di-GMP, c-di-AMP and c-AMP-GMP using the cyclic-AMP-GMP synthetase, DncV. The enzymatic reaction efficiently produced most cyclic dinucleotide analogs, such as 2'-amino sugar substitutions and phosphorothioate backbone modifications, for all three types of cyclic dinucleotides without the use of protecting groups or organic solvents. We used these novel analogs to explore differences in phosphate backbone and 2'-hydroxyl recognition between GEMM-I and GEMM-Ib riboswitches.