A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration

Manganese (Mn)-sensing riboswitches protect bacteria from Mn toxicity by upregulating expression of Mn exporters. The Mn aptamers share key features but diverge in other important elements, including within the metal-binding core. Although X-ray crystal structures of isolated aptamers exist, these structural snapshots lack crucial details about how the aptamer communicates the presence or absence of ligand to the expression platform. In this work, we investigated the Mn-sensing translational riboswitches inE. coli(mntPandalx), which differ in aptamer secondary structure, nucleotide sequence, and pH-dependence of Mn response. We performed co-transcriptional RNA chemical probing, allowing us to visualize RNA folding intermediates that form and resolveen routeto the final folded riboswitch. For the first time, we report that sampling of metal ions by the RNA begins before the aptamer synthesis and folding are complete. At a single-nucleotide resolution, we pinpoint the transcription window where “riboswitching” occurs in response to Mn binding and uncover key differences in how thealxandmntPriboswitches fold. Finally, we describe riboswitch-specific effects of pH, providing insights into how two members of the same riboswitch family differentially sense two distinct environmental cues: concentration of Mn and pH.

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Opportunities for Riboswitch Inhibition by Targeting Co-Transcriptional RNA Folding Events

Stephen,

Palmer,

Mishanina

2024

IJMS

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Antibiotic resistance is a critical global health concern, causing millions of prolonged bacterial infections every year and straining our healthcare systems. Novel antibiotic strategies are essential to combating this health crisis and bacterial non-coding RNAs are promising targets for new antibiotics. In particular, a class of bacterial non-coding RNAs called riboswitches has attracted significant interest as antibiotic targets. Riboswitches reside in the 5′-untranslated region of an mRNA transcript and tune gene expression levels in cis by binding to a small-molecule ligand. Riboswitches often control expression of essential genes for bacterial survival, making riboswitch inhibitors an exciting prospect for new antibacterials. Synthetic ligand mimics have predominated the search for new riboswitch inhibitors, which are designed based on static structures of a riboswitch’s ligand-sensing aptamer domain or identified by screening a small-molecule library. However, many small-molecule inhibitors that bind an isolated riboswitch aptamer domain with high affinity in vitro lack potency in vivo. Importantly, riboswitches fold and respond to the ligand during active transcription in vivo. This co-transcriptional folding is often not considered during inhibitor design, and may explain the discrepancy between a low Kd in vitro and poor inhibition in vivo. In this review, we cover advances in riboswitch co-transcriptional folding and illustrate how intermediate structures can be targeted by antisense oligonucleotides—an exciting new strategy for riboswitch inhibitor design.

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A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration

Cited by 3 publications

References 79 publications

Beyond ligand binding: Single molecule observation reveals how riboswitches integrate multiple signals to balance bacterial gene regulation

Beyond ligand binding: Single molecule observation reveals how riboswitches integrate multiple signals to balance bacterial gene regulation

Structurally distinct manganese-sensing riboswitch aptamers regulate diverse expression platform architectures

Opportunities for Riboswitch Inhibition by Targeting Co-Transcriptional RNA Folding Events

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