Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic). While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a co-transcriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms.

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.

The lysine riboswitch is associated to the lysC gene in Bacillus subtilis, and the binding of lysine modulates the RNA structure to allow the formation of an intrinsic terminator presumably involved in transcription attenuation. The complex secondary structure of the lysine riboswitch aptamer is organized around a five-way junction that undergoes structural changes upon ligand binding. Using single-round transcription assays, we show that a loop-loop interaction is important for lysine-induced termination of transcription. Moreover, upon close inspection of the secondary structure, we find that an unconventional kinkturn motif is present in one of the stems participating in the loop-loop interaction. We show that the K-turn adopts a pronounced kink and that it binds the K-turn-binding protein L7Ae of Archaeoglobus fulgidus in the low nanomolar range. The functional importance of this K-turn motif is revealed from single-round transcription assays, which show its importance for efficient transcription termination. This motif is essential for the loop-loop interaction, and consequently, for lysine binding. Taken together, our results depict for the first time the importance of a K-turn-dependent loop-loop interaction for the transcription regulation of a lysine riboswitch.

The Bacillus subtilis lysC lysine riboswitch modulates its own gene expression upon lysine binding through a transcription attenuation mechanism. The riboswitch aptamer is organized around a single five-way junction that provides the scaffold for two long-range tertiary interactions (loop L2–loop L3 and helix P2–loop L4)—all of this for the creation of a specific lysine binding site. We have determined that the interaction P2–L4 is particularly important for the organization of the ligand-binding site and for the riboswitch transcription attenuation control. Moreover, we have observed that a folding synergy between L2–L3 and P2–L4 allows both interactions to fold at lower magnesium ion concentrations. The P2–L4 interaction is also critical for the close juxtaposition involving stems P1 and P5. This is facilitated by the presence of lysine, suggesting an active role of the ligand in the folding transition. We also show that a previously uncharacterized stem–loop located in the expression platform is highly important for the riboswitch activity. Thus, folding elements located in the aptamer and the expression platform both influence the lysine riboswitch gene regulation.

Objectives: Maternal satisfaction during the birthing process has been well documented, whereas little is known about the fathers' birth experiences. Our objective was to evaluate and compare the birth satisfaction of mothers and fathers.