Comprehensive mutational analysis of the sequence-function relationship within a viral internal ribosome entry site.

The cricket paralysis virus (CrPV) intergenic region internal ribosome entry site (IRES) binds to the ribosome without the need for any initiation factors. Their length, simple mechanism, and ability to function in diverse cell-free systems make CrPV-like IRESs useful tools to study the mechanism of translation and to express proteins. We report the use of a RelE-based next-generation sequencing method, termed SMARTI (sequencing-based mutational analysis of RNA translation initiation), to quantitatively determine the function of over 81 000 single and double mutants of CrPV IRES.

Decoding the Complex Functional Landscape of the Riboswitches.

The class is the most diverse riboswitch class to date, recognizing structurally and chemically diverse ligands using only minor changes in sequence and structure. Structural studies have demonstrated how sequence changes correspond to altered specificity; however, they are insufficient to define the requirements for functional riboswitch specificity. Here, we report an extensive mutational analysis of the ppGpp riboswitch to investigate the functional role in transcriptional control for this variant riboswitch.

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.

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.

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.

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.

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.

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.

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.

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.

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

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 .

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

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 p.

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 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.

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.

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 were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer.

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 resulted in over 1000-fold increased fluoride sensitivity.

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.

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

Methylobacterium radiotolerans

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

Two classes of riboswitches related to the 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.

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.

Structural basis for ligand binding to the guanidine-II riboswitch.

The guanidine-II riboswitch, also known as , is a conserved mRNA element with more than 800 examples in bacteria. It consists of two stem-loops capped by identical, conserved tetraloops that are separated by a linker region of variable length and sequence. Like the guanidine-I riboswitch, it controls the expression of guanidine carboxylases and SugE-like genes.

Fluoride export (FEX) proteins from fungi, plants and animals are 'single barreled' channels containing one functional and one vestigial ion pore.

The fluoride export protein (FEX) in yeast and other fungi provides tolerance to fluoride (F-), an environmentally ubiquitous anion. FEX efficiently eliminates intracellular fluoride that otherwise would accumulate at toxic concentrations. The FEX homolog in bacteria, Fluc, is a 'double-barreled' channel formed by dimerization of two identical or similar subunits.

Mycofumigation through production of the volatile DNA-methylating agent -methyl--nitrosoisobutyramide by fungi in the genus .

Antagonistic microorganisms produce antimicrobials to inhibit the growth of competitors. Although water-soluble antimicrobials are limited to proximal interactions via aqueous diffusion, volatile antimicrobials are able to act at a distance and diffuse through heterogeneous environments. Here, we identify the mechanism of action of , an endophytic fungus known for its volatile antimicrobial activity toward a wide range of human and plant pathogens and its potential use in mycofumigation.