Riboswitches are commonly used by bacteria to detect a variety of metabolites and ions to regulate gene expression. To date, nearly 40 different classes of riboswitches have been discovered, experimentally validated, and modeled at atomic resolution in complex with their cognate ligands. The research findings produced since the first riboswitch validation reports in 2002 reveal that these noncoding RNA domains exploit many different structural features to create binding pockets that are extremely selective for their target ligands. Some riboswitch classes are very common and are present in bacteria from nearly all lineages, whereas others are exceedingly rare and appear in only a few species whose DNA has been sequenced. Presented herein are the consensus sequences, structural models, and phylogenetic distributions for all validated riboswitch classes. Based on our findings, we predict that there are potentially many thousands of distinct bacterial riboswitch classes remaining to be discovered, but that the rarity of individual undiscovered classes will make it increasingly difficult to find additional examples of this RNA-based sensory and gene control mechanism.

SUMMARY The guanidyl moiety is a component of fundamental metabolites including the amino acid arginine, the energy carrier creatine, and the nucleobase guanine. Curiously, reports regarding the importance of free guanidine in biology are sparse and no biological receptors that specifically recognize this compound have been previously identified. We report that many members of the ykkC motif RNA, the longest-unresolved riboswitch candidate, naturally sense and respond to guanidine. This RNA is found throughout much of the bacterial domain of life, where it commonly controls the expression of proteins annotated as urea carboxylases and multidrug efflux pumps. Our analyses reveal that these proteins likely function as guanidine carboxylases and guanidine transporters, respectively. Furthermore, we demonstrate that bacteria are capable of endogenously producing guanidine. These and related findings demonstrate that free guanidine is a biologically relevant compound and several gene families exists that can alleviate guanidine toxicity.

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.

Recently, it was determined that representatives of the riboswitch candidates called ykkC and ykkC-III directly bind free guanidine. Guanidine-binding ykkC motif RNAs, now renamed guanidine-I riboswitches, were demonstrated to commonly regulate the expression of genes encoding guanidine carboxylases, as well as others encoding guanidine efflux proteins such as EmrE and SugE. Likewise, genes encoding similar efflux proteins are associated with ykkC-III motif RNAs, which have now been renamed guanidine-III riboswitches. Prior to the validation of guanidine as the ligand for these newly-established riboswitch classes, another RNA motif was discovered by comparative genomic analysis and termed mini-ykkC due to its small size and gene associations similar to the original ykkC motif. It was hypothesized that these distinct RNA structures might respond to the same ligand. However, the small size and repetitive nature of mini-ykkC RNAs suggested that it might respond to ligand via the action of a protein factor. Herein we demonstrate that, despite its extremely simple architecture, mini-ykkC motif RNAs constitute a distinct class of guanidine-sensing RNAs, called guanidine-II riboswitches. Surprisingly, each of the two stem loops that comprise the mini-ykkC motif appears to directly bind free guanidine in a cooperative manner. These findings reveal that bacteria make extensive use of diverse guanidine-responsive riboswitches to overcome the toxic effects of this compound.

SignificanceBacteria and other organisms make extensive use of signaling molecules that are derived from ribonucleotides or their derivatives. Previously, five riboswitch classes had been discovered that sense the four RNA-derived signaling molecules: c-di-GMP, c-di-AMP, c-AMP-GMP, and ZTP. We now report the discovery and biochemical validation of bacterial riboswitches for the widespread alarmone guanosine tetraphosphate (ppGpp), which signals metabolic and physiological adaptations to starvation. These findings expand the number of natural partnerships between riboswitches and ribonucleotide-like signaling molecules, and provide RNA-based sensors for detecting ppGpp production in cells.