Enzymes made of RNA catalyze reactions that are essential for protein synthesis and RNA processing. However, such natural ribozymes are exceedingly rare, as evident by the fact that the discovery rate for new classes has dropped to one per decade from about one per year during the 1980s. Indeed, only 11 distinct ribozyme classes have been experimentally validated to date. Recently, we recognized that self-cleaving ribozymes frequently associate with certain types of genes from bacteria. Herein this synteny was exploited to identify divergent architectures for two previously known ribozyme classes and to discover additional noncoding RNA motifs that are self-cleaving RNA candidates. Three new self-cleaving classes, named twister sister, pistol and hatchet, have been identified from this collection, suggesting that even more ribozymes remain hidden in modern cells.
Fluorine is an abundant element and is toxic to organisms from bacteria to humans, but the mechanisms by which eukaryotes resist fluoride toxicity are unknown. The Escherichia coli gene crcB was recently shown to be regulated by a fluoride-responsive riboswitch, implicating it in fluoride response. There are >8,000 crcB homologs across all domains of life, indicating that it has an important role in biology. Here we demonstrate that eukaryotic homologs [renamed FEX (fluoride exporter)] function in fluoride export. FEX KOs in three eukaryotic model organisms, Neurospora crassa, Saccharomyces cerevisiae, and Candida albicans, are highly sensitized to fluoride (>200-fold) but not to other halides. Some of these KO strains are unable to grow in fluoride concentrations found in tap water. Using the radioactive isotope of fluoride, 18 F, we developed an assay to measure the intracellular fluoride concentration and show that the FEX deletion strains accumulate fluoride in excess of the external concentration, providing direct evidence of FEX function in fluoride efflux. In addition, they are more sensitive to lower pH in the presence of fluoride. These results demonstrate that eukaryotic FEX genes encode a previously unrecognized class of fluoride exporter necessary for survival in standard environmental conditions. toxicity resistance | environmental toxin | ion transport | dual membrane topology
Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min −1 under physiological Mg 2+ and pH conditions. The reaction proceeds via the nucleophilic attack of a 2 ′ -oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA.
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