A variant of guanidine-IV riboswitches exhibits evidence of a distinct ligand specificity.

Riboswitches are regulatory RNAs that specifically bind a small molecule or ion. Like metabolite-binding proteins, riboswitches can evolve new ligand specificities, and some examples of this phenomenon have been validated. As part of work based on comparative genomics to discover novel riboswitches, we encountered a candidate riboswitch with striking similarities to the recently identified guanidine-IV riboswitch. This candidate riboswitch, the Gd4v motif, is predicted in four distinct bacterial phyla, thus almost as widespread as the guanidine-IV riboswitch. Bioinformatic and experimental analysis suggest that the Gd4v motif is a riboswitch that binds a ligand other than guanidine. It is found associated with gene classes that differ from genes regulated by confirmed guanidine riboswitches. In inline-probing assays, we showed that free guanidine binds only weakly to one of the tested sequences of the variant. Further tested compounds did not show binding, attenuation of transcription termination, or activation of a genetic reporter construct. We characterized an N-acetyltransferase frequently associated with the Gd4v motif and compared its substrate preference to an N-acetyltransferase that occurs under control of guanidine-IV riboswitches. The substrates of this Gd4v-motif-associated enzyme did not show activity for Gd4v RNA binding or transcription termination. Hence, the ligand of the candidate riboswitch motif remains unidentified. The variant RNA motif is predominantly found in gut metagenome sequences, hinting at a ligand that is highly relevant in this environment. This finding is a first step to determining the identity of this unknown ligand, and understanding how guanidine-IV-riboswitch-like structures can evolve to bind different ligands.

Widespread bacterial utilization of guanidine as nitrogen source.

Guanidine is sensed by at least four different classes of riboswitches that are widespread in bacteria. However, only very few insights into physiological roles of guanidine exist. Genes predominantly regulated by guanidine riboswitches are Gdx transporters exporting the compound from the bacterial cell. In addition, urea/guanidine carboxylases and associated hydrolases and ABC transporters are often found combined in guanidine-inducible operons. We noted that the associated ABC transporters are configured to function as importers, challenging the current view that riboswitches solely control the detoxification of guanidine in bacteria. We demonstrate that the carboxylase pathway enables utilization of guanidine as sole nitrogen source. We isolated three enterobacteria (Raoultella terrigena, Klebsiella michiganensis, and Erwinia rhapontici) that utilize guanidine efficiently as N-source. Proteome analyses show that the expression of a carboxylase, associated hydrolases and transport genes is strongly induced by guanidine. Finding two urea/guanidine carboxylase enzymes in E. rhapontici, we demonstrate that the riboswitch-controlled carboxylase displays specificity toward guanidine, whereas the other enzyme prefers urea. We characterize the distribution of riboswitch-associated carboxylases and Gdx exporters in bacterial habitats by analyzing available metagenome data. The findings represent a paradigm shift from riboswitch-controlled detoxification of guanidine to the uptake and assimilation of this enigmatic nitrogen-rich compound.

Discovery and characterization of a fourth class of guanidine riboswitches.

Riboswitches are RNAs that specifically sense a small molecule and regulate genes accordingly. The recent discovery of guanidine-binding riboswitches revealed the biological significance of this compound, and uncovered genes related to its biology. For example, certain sugE genes encode guanidine exporters and are activated by the riboswitches to reduce toxic levels of guanidine in the cell. In order to study guanidine biology and riboswitches, we applied a bioinformatics strategy for discovering additional guanidine riboswitches by searching for new candidate motifs associated with sugE genes. Based on in vitro and in vivo experiments, we determined that one of our six best candidates is a new structural class of guanidine riboswitches. The expression of a genetic reporter was induced 80-fold in response to addition of 5 mM guanidine in Staphylococcus aureus. This new class, called the guanidine-IV riboswitch, reveals additional guanidine-associated protein domains that are extremely rarely or never associated with previously established guanidine riboswitches. Among these protein domains are two transporter families that are structurally distinct from SugE, and could represent novel types of guanidine exporters. These results establish a new metabolite-binding RNA, further validate a bioinformatics method for finding riboswitches and suggest substrate specificities for as-yet uncharacterized transporter proteins.

A Theophylline-Responsive Riboswitch Regulates Expression of Nuclear-Encoded Genes.

Riboswitches are small cis-regulatory RNA elements that regulate gene expression by conformational changes in response to ligand binding. Synthetic riboswitches have been engineered as versatile and innovative tools for gene regulation by external application of their ligand in prokaryotes and eukaryotes. In plants, synthetic riboswitches were used to regulate gene expression in plastids, but the application of synthetic riboswitches for the regulation of nuclear-encoded genes in planta remains to be explored. Here, we characterize the properties of a theophylline-responsive synthetic aptazyme for control of nuclear-encoded transgenes in Arabidopsis (Arabidopsis thaliana). Activation of the aptazyme, inserted in the 3' UTR of the target gene, resulted in rapid self-cleavage and subsequent decay of the mRNA. This riboswitch allowed reversible, theophylline-dependent down-regulation of the GFP reporter gene in a dose- and time-dependent manner. Insertion of the riboswitch into the ONE HELIX PROTEIN1 gene allowed complementation of ohp1 mutants and induction of the mutant phenotype by theophylline. GFP and ONE HELIX PROTEIN1 transcript levels were downregulated by up to 90%, and GFP protein levels by 95%. These results establish artificial riboswitches as tools for externally controlled gene expression in synthetic biology in plants or functional crop design.