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.
The nadA motif is a riboswitch candidate present in various Acidobacteria species that was previously identified by bioinformatic analysis of bacterial DNA data sets. More than 100 unique representatives have been identified exclusively upstream of nadA genes, which code for an enzyme in the biosynthetic pathway of the ubiquitous coenzyme NAD + . The architecture of nadA motif RNAs suggests they use structurally similar tandem ligand-binding aptamer domains to control translation initiation. Biochemical analyses reveal that the first domain selectively binds ligands carrying an adenosine 5 ′ ′ ′ ′ ′ -diphosphate (5 ′ ′ ′ ′ ′ ADP) moiety, including NAD + and its reduced form, NADH. Genetic analyses indicate that a tandem nadA motif RNA suppresses gene expression when NAD + is abundant, and that both aptamer domains are required for maximal gene regulation. However, we have not observed selective binding of the nicotinamide moiety of NAD + or binding by the second putative aptamer in vitro, despite sequence and structural similarities between the tandem domains.
The experimental validation of three distinct riboswitch classes has revealed that many bacterial cells naturally produce guanidine, and that living systems have evolved a variety of genes involved in the metabolism and transport of this toxic metabolite. There are numerous biochemical curiosities and mysteries that spring from these advances, which will make for interesting research topics in the coming years.
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