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Several structured noncoding RNAs in bacteria are essential contributors to fundamental cellular processes. Thus, discoveries of additional ncRNA classes provide opportunities to uncover and explore biochemical mechanisms relevant to other major and potentially ancient processes. A candidate structured ncRNA named the “raiA motif” has been found via bioinformatic analyses in over 2,500 bacterial species. The gene coding for the RNA typically resides between the raiA and comFC genes of many species of Bacillota and Actinomycetota. Structural probing of the raiA motif RNA from the Gram-positive anaerobe Clostridium acetobutylicum confirms key features of its sophisticated secondary structure model. Expression analysis of raiA motif RNA reveals that the RNA is constitutively produced but reaches peak abundance during the transition from exponential growth to stationary phase. The raiA motif RNA becomes the fourth most abundant RNA in C. acetobutylicum, excluding ribosomal RNAs and transfer RNAs. Genetic disruption of the raiA motif RNA causes cells to exhibit substantially decreased spore formation and diminished ability to aggregate. Restoration of normal cellular function in this knock-out strain is achieved by expression of a raiA motif gene from a plasmid. These results demonstrate that raiA motif RNAs normally participate in major cell differentiation processes by operating as a trans-acting factor. [ABSTRACT FROM AUTHOR]

Riboswitches rely on structured aptamer domains to selectively sense their target ligands and regulate gene expression. However, some riboswitch aptamers in bacteria carry mutations in their otherwise strictly conserved binding pockets that change ligand specificities. The aptamer domain of a riboswitch class originally found to selectively sense guanine forms a three-stem junction that has since been observed to exploit numerous alterations in its ligand-binding pocket. These rare variants have modified their ligand specificities to sense other purines or purine derivatives, including adenine, 2'-deoxyguanosine (three classes), and xanthine. Herein, we report the characteristics of a rare variant that is narrowly distributed in the Paenibacillaceae family of bacteria. Known representatives are always associated with genes encoding 8-oxoguanine deaminase. As predicted from this gene association, these variant riboswitches tightly bind 8-oxoguanine (8-oxoG), strongly discriminate against other purine derivatives, and function as genetic "ON" switches. Following exposure of cells to certain oxidative stresses, a representative 8-oxoG riboswitch activates gene expression, likely caused by the accumulation of 8-oxoG due to oxidative damage to G nucleobases in DNA, RNA, and the nucleotide pool. Furthermore, an engineered version of the variant aptamer was prepared that exhibits specificity for 8-oxoadenine, further demonstrating that RNA aptamers can acquire mutations that expand their ability to detect and respond to oxidative damage. [ABSTRACT FROM AUTHOR]

Several mRNA aptamers have been identified in Mesoplasma florum that have sequence and structural features resembling those of guanine and adenine riboswitches. Two features distinguish these RNAs from established purine-sensing riboswitches. All possess shortened hairpin-loop sequences expected to alter tertiary contacts known to be critical for aptamer folding. The RNAs also carry nucleotide changes in the core of each aptamer that otherwise is strictly conserved in guanine and adenine riboswitches. Some aptamers retain the ability to selectively bind guanine or adenine despite these mutations. However, one variant type exhibits selective and high-affinity binding of 2'-deoxyguanosine, which is consistent with its occurrence in the 5' untranslated region of an operon containing ribonucleotide reductase genes. The identification of riboswitch variants that bind nucleosides and reject nucleobases reveals that natural metabolite-sensing RNA motifs can accrue mutations that expand the diversity of ligand detection in bacteria. allosteric RNA | aptamer | metabolite | ribonucleotide reductase | transcription termination

We have discovered a large and highly conserved RNA motif that typically resides in a noncoding section of a multigene messenger RNA in extremophilic Gram-positive eubacteria. RNAs of this class adopt an ornate secondary structure, are large compared with most other noncoding RNAs, and have been identified only in certain extremophilic bacteria. These ornate, large, extremophilic (OLE) RNAs have a length of [approximately equal to] 610 nucleotides, and the 35 representatives examined exhibit extraordinary conservation of nucleotide sequence and base pairing. Structural probing of the OLE RNA from Bacillus halodurans corroborates a complex secondary structure model predicted from comparative sequence analysis. The patterns of structural conservation, and its unique phylogenetic distribution, suggest that OLE RNA carries out a complex and critical function only in certain extremophilic bacteria. isoprenoid | riboswitch | ribozyme | superoperon

The expression of certain genes involved in fundamental metabolism is regulated by metabolite-binding 'riboswitch' elements embedded within their corresponding mRNAs. We have identified at least six additional elements within the Bacillus subtilis genome that exhibit characteristics of riboswitch function (glms, gcvT, ydaO/yuaA, ykkC/yxkD, ykoK, and yybP/ykoY). These motifs exhibit extensive sequence and secondary-structure conservation among many bacterial species and occur upstream of related genes. The element located upstream of the glms gene in Gram-positive organisms functions as a metabolite-dependent ribozyme that responds to glucosamine-6-phosphate. Other motifs form complex folded structures when transcribed as RNA molecules and carry intrinsic terminator structures. These findings indicate that riboswitches serve as a major genetic regulatory mechanism for the control of metabolic genes in many microbial species.

The aptamer portions of previously reported riboswitch classes that sense guanine, adenine, or 2'-deoxyguanosine are formed by a highly similar three-stem junction with distinct nucleotide sequences in the regions joining the stems. The nucleotides in these joining regions form the major features of the selective ligand-binding pocket for each aptamer. Previously, we reported the existence of additional, rare variants of the predominant guanine-sensing riboswitch class that carry nucleotide differences in the ligand-binding pocket, suggesting that these RNAs have further diversified their structures and functions. Herein, we report the discovery and analysis of three naturally occurring variants of guanine riboswitches that are narrowly distributed across Firmicutes. These RNAs were identified using comparative sequence analysis methods, which also revealed that some of the gene associations for these variants are atypical for guanine riboswitches or their previously known natural variants. Binding assays demonstrate that the newfound variant riboswitch representatives recognize xanthine, guanine, or 20 -deoxyguanosine, with the guanine class exhibiting greater discrimination against related purines than the more common guanine riboswitch class reported previously. These three additional variant classes, together with the four previously discovered riboswitch classes that employ the same three-stem junction architecture, reveal how a simple structural framework can be diversified to expand the range of purine-based ligands sensed by RNA. [ABSTRACT FROM AUTHOR]

Natural ribozymes require metal ion cofactors that aid both in structural folding and in chemical catalysis. In contrast, many protein enzymes produce dramatic rate enhancements using only the chemical groups that are supplied by their constituent amino acids. This fact is widely viewed as the most important feature that makes protein a superior polymer for the construction of biological catalysts. Herein we report the in vitro selection of a catalytic DNA that uses histidine as an active component for an RNA cleavage reaction. An optimized deoxyribozyme from this selection requires L-histidine or a closely related analog to catalyze RNA phosphoester cleavage, producing a rate enhancement of [approximately equal to]1-million-fold over the rate of substrate cleavage in the absence of enzyme. Kinetic analysis indicates that a DNA-histidine complex may perform a reaction that is analogous to the first step of the proposed catalytic mechanism of RNase A, in which the imidazole group of histidine serves as a general base catalyst. Similarly, ribozymes of the 'RNA world' may have used amino acids and other small organic cofactors to expand their otherwise limited catalytic potential.