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.

Transfer RNAs (tRNAs) are central players in translation, functioning as adapter molecules between the informational level of nucleic acids and the functional level of proteins. They show a highly conserved secondary and tertiary structure and the highest density of post-transcriptional modifications among all RNAs. These modifications concentrate in two hotspots—the anticodon loop and the tRNA core region, where the D- and T-loop interact with each other, stabilizing the overall structure of the molecule. These modifications can cause large rearrangements as well as local fine-tuning in the 3D structure of a tRNA. The highly conserved tRNA shape is crucial for the interaction with a variety of proteins and other RNA molecules, but also needs a certain flexibility for a correct interplay. In this context, it was shown that tRNA modifications are important for temperature adaptation in thermophilic as well as psychrophilic organisms, as they modulate rigidity and flexibility of the transcripts, respectively. Here, we give an overview on the impact of modifications on tRNA structure and their importance in thermal adaptation.

The discovery of structured non-coding RNAs (ncRNAs) in bacteria can reveal new facets of biology and biochemistry. Comparative genomics analyses executed by powerful computer algorithms have successfully been used to uncover many novel bacterial ncRNA classes in recent years. However, this general search strategy favors the discovery of more common ncRNA classes, whereas progressively rarer classes are correspondingly more difficult to identify. In the current study, we confront this problem by devising several methods to select subsets of intergenic regions that can concentrate these rare RNA classes, thereby increasing the probability that comparative sequence analysis approaches will reveal their existence. By implementing these methods, we discovered 224 novel ncRNA classes, which include ROOL RNA, an RNA class averaging 581 nt and present in multiple phyla, several highly conserved and widespread ncRNA classes with properties that suggest sophisticated biochemical functions and a multitude of putative cis-regulatory RNA classes involved in a variety of biological processes. We expect that further research on these newly found RNA classes will reveal additional aspects of novel biology, and allow for greater insights into the biochemistry performed by ncRNAs.

Major changes in bacterial physiology including biofilm and spore formation involve signaling by the cyclic dinucleotides c-di-GMP and c-di-AMP. Recently, another second messenger dinucleotide, c-AMP-GMP, was found to control chemotaxis and colonization by Vibrio cholerae. We have identified a superregulon of genes controlled by c-AMP-GMP in numerous Deltaproteobacteria, including Geobacter species that use extracellular insoluble metal oxides as terminal electron acceptors. This exoelectrogenic process has been studied for its possible utility in energy production and bioremediation. Many genes involved in adhesion, pilin formation, and others that are important for exoelectrogenesis are controlled by members of a variant riboswitch class that selectively bind c-AMP-GMP. These RNAs constitute, to our knowledge, the first known specific receptors for c-AMP-GMP and reveal that this molecule is used by many bacteria to control specialized physiological processes.

The glmS-riboswitch is unique among riboswitch families as it represents a metabolite-dependent ribozyme that undergoes self-cleavage upon recognition of glucosamin-6-phosphate. The glmS-riboswitch is located in the 5'-untranslated region of bacterial genes involved in cell wall biosynthesis. Therefore, this riboswitch represents a promising target for developing new antibiotics. We describe the metabolite-dependent glmS-riboswitch of pathologically relevant and vancomycin-resistant Staphylococcus aureus and the discovery and synthesis of a carba-sugar with potency similar to that of the native metabolite glucosamine-6-phosphate in modulating riboswitch activity. This compound represents a valuable lead structure for the development of antibiotics with a novel mode of action.

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.

Riboswitches are RNAs that form complex, folded structures that selectively bind small molecules or ions. As with certain groups of protein enzymes and receptors, some riboswitch classes have evolved to change their ligand specificity. We developed a procedure to systematically analyze known riboswitch classes to find additional variants that have altered their ligand specificity. This approach uses multiple-sequence alignments, atomic-resolution structural information, and riboswitch gene associations. Among the discoveries are unique variants of the guanine riboswitch class that most tightly bind the nucleoside 2′-deoxyguanosine. In addition, we identified variants of the glycine riboswitch class that no longer recognize this amino acid, additional members of a rare flavin mononucleotide (FMN) variant class, and also variants of c-di-GMP-I and -II riboswitches that might recognize different bacterial signaling molecules. These findings further reveal the diverse molecular sensing capabilities of RNA, which highlights the potential for discovering a large number of additional natural riboswitch classes.iboswitches are structured noncoding RNA domains that regulate gene expression in response to the selective binding of small-molecule or ion ligands. The discovery of numerous classes of riboswitches has helped reveal how RNAs can form exquisitely precise ligand-binding pockets using only the four common RNA nucleotides (1-4). Furthermore, each discovery links the riboswitch ligand to the protein products of the genes under regulation. Recent riboswitch findings have exposed unique facets of biology, such as the widespread molecular mechanisms that confer fluoride (5) or guanidine (6) resistance, that maintain metal ion homeostasis (7-9), and that control important bacterial processes such as sporulation, biofilm formation, and chemotaxis (10-14). Thus, the identification of additional riboswitch classes promises to offer insights into otherwise hidden biological processes and their regulation.Riboswitch variants have been reported previously, wherein the ligand-binding "aptamer" domain has mutated to accommodate a different metabolite or signaling compound. The identification of such RNAs provides rare opportunities to study how small changes in RNA sequence can lead to major changes in smallmolecule ligand affinity. There have been seven examples, either experimentally validated or suspected, of ligand specificity changes reported to date. These include guanine aptamer variations present in riboswitches for adenine (15) and 2′-deoxyguanosine (2′-dG) (16), c-di-GMP-I aptamer variations that result in riboswitches that bind the recently discovered bacterial signaling molecule c-AMP-GMP (13, 14), and coenzyme B 12 aptamer changes (17, 18) that yield riboswitches selective for aquocobalamin (19). Three additional ligand specificity changes are suspected. Namely, some molybdenum cofactor riboswitches appear to exploit an altered aptamer structure to selectively recognize tungsten cofactor (20), certain flavin mononu...

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