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Riboswitches are cis-acting regulatory RNAs that are found in the 5ʹ untranslated region (UTR) of the genes they regulate. These elements modulate gene expression by structural rearrangements in response to a wide variety of physiological signals. The S-adenosylmethionine (SAM)-responsive S-box RNAs are one of the most prevalent classes of riboswitches. S-box riboswitches are found primarily in Firmicutes and regulate the expression of genes involved in methionine and SAM metabolism. These riboswitches consist of an aptamer domain that binds SAM and an expression platform that undergoes a SAM-dependent conformational rearrangement to regulate the expression of downstream coding regions. When the intracellular level of SAM is high, SAM binds to the aptamer domain and stabilizes a terminator helix to cause premature termination of transcription. When the SAM level is low, a competing antiterminator helix forms, which allows transcription of the downstream genes. Bioinformatic analyses have suggested that a rarer class of S-box riboswitches may regulate gene expression at the level of translation initiation in some non-Firmicutes. Indeed, in this study, we identified a translational S-box element in the metI gene of Desulfurispirillum indicum. We demonstrate that this RNA undergoes SAM-dependent changes in the structure of the expression platform and the efficiency of ribosome binding, which are consistent with the regulation at the level of translation initiation. We hypothesized that the translational and transcriptional S-box riboswitches that are located in the same gene in different organisms would regulate expression using different regulatory strategies. Regulatory mechanisms of riboswitches have been shown to be influenced by the kinetics of ligand interaction. A major goal of this work was to compare the ligand binding properties of the transcriptional and translational metI S-box riboswitches. We show that despite the structural similarity, the transcriptional and translational S-box riboswitches have significantly different ligand dissociation rates, suggesting that the molecular regulatory mechanisms might be different between the two riboswitches. Additionally, we identified an internal loop in the variable region of the S-box aptamer domain that contributes to these differences by influencing the flexibility of residues in the SAM-binding pocket, and therefore affects the molecular mechanism of riboswitch function. The identification and characterization of the translational variants expands our current knowledge of the diversity that exists in the S-box riboswitch regulatory mechanisms. Moreover, this work adds to the few studies that have directly compared the properties of transcriptional and translational riboswitches from the same class. The elements that are close to the ligand-binding pocket could also contribute to the regulatory differences among the members of other riboswitch classes. Overall, this work advances our understanding of the S-box riboswitch mechanism and could provide insights into understanding the regulatory mechanisms of other riboswitches. |
