Biochemical Validation of a Fourth Guanidine Riboswitch Class in Bacteria

Abstract

An intriguing consequence of ongoing riboswitch discovery efforts is the occasional identification of metabolic or toxicity response pathways for unusual ligands. Recently, we reported the experimental validation of three distinct bacterial riboswitch classes that regulate gene expression in response to the selective binding of a guanidinium ion. These riboswitch classes, called guanidine-I, -II, and -III, regulate numerous genes whose protein products include previously misannotated guanidine exporters and enzymes that degrade guanidine via an initial carboxylation reaction. Guanidine is now recognized as the primal substrate of many multidrug efflux pumps that are important for bacterial resistance to certain antibiotics. Guanidine carboxylase enzymes had long been annotated as urea carboxylase enzymes but are now understood to participate in guanidine degradation. Herein, we report the existence of a fourth riboswitch class for this ligand, called guanidine-IV. Members of this class use a novel aptamer to selectively bind guanidine and use an unusual expression platform arrangement that is predicted to activate gene expression when ligand is present. The wide distribution of this abundant riboswitch class, coupled with the striking diversity of other guanidine-sensing RNAs, demonstrates that many bacterial species maintain sophisticated sensory and genetic mechanisms to avoid guanidine toxicity. This finding further highlights the mystery regarding the natural source of this nitrogen-rich chemical moiety.N umerous riboswitch classes 1−3 for RNA-derived com-

Characterization of Structure and Dynamics of the Guanidine‐II Riboswitch from Escherichia coli by NMR Spectroscopy and Small‐Angle X‐ray Scattering (SAXS)

“…We noted that U17 shows larger changes than G9 and G10 but we cannot explain this. Upon addition of Gdm + (up to 8 mM), the formation of two GC kissing base pairs is reasoned by the detection of two new imino proton signals (Figure 3C 2D spectra ), in line with the base pairs observed in crystal structures [19] . For Gdn23 wt and Gdn49 wt, we see shifts both, for Mg 2+ and additional shift of Gdm + interaction.…”

“…[18] To date, four different classes of Gdm + -sensing riboswitches have been discovered termed guanidine-I to guanidine-IV. [15,18,19] Many of these Gdm + -sensing riboswitches are biologically well characterized and structural models of isolated hairpins were reported for all the four riboswitch classes including Gdn-II from Escherichia coli (PDB: 5NDI), from Gloeobacter violaceus (5NOM and 5NDH) and from Pseudomonas aeruginosa (5VJ9). [14,20] The structures reveal striking similarities of ligand binding pockets between these different riboswitches.…”

Comprehensive discovery of novel structured noncoding RNAs in 26 bacterial genomes

“…Structurally, the HRC-8-4 motif vaguely resembles the recently validated HMP-PP [ 17 ] and guanidine-IV [ 21 , 22 ] riboswitch classes. Like these experimentally validated ribo-switch classes, the HRC-8-4 motif generally conforms to the classic architecture of an intrinsic terminator stem [ 64–66 ], which includes a strong stem followed by a run of U nucleotides.…”

“…HMP-PP and guanidine-IV riboswitches appear to exploit these conserved nucleotides to form the aptamers for their target ligands. This precludes the formation of the terminator stem when ligands bind and thus they function as genetic ‘ON’ switches [ 17 , 21 , 22 ]. We speculate that the HRC-8-4 motif might perform a similar function, although we again cannot be certain that the ligand is a small molecule.…”

Combining Coarse-Grained Simulations and Single Molecule Analysis Reveals a Three-State Folding Model of the Guanidine-II Riboswitch

“…So far, four classes of riboswitches have been identified that bind the cationic molecule guanidinium (Gdm + ): guanidine-I ( Breaker et al, 2017 ), -II ( Sherlock et al, 2017 ), -III ( Sherlock and Breaker 2017 ) and -IV ( Salvail et al, 2020 ). The corresponding genes are in most cases involved in Gdm + detoxification, and code for proteins like guanidine carboxylases or multidrug efflux pumps (e. g., SugE).…”