# Regulatory helix plays a key role in genetic ON–OFF switching for the 2′-deoxyguanosine-sensing mRNA element

## TL;DR

This study reveals how a specific mRNA element regulates gene expression by sensing a metabolite during RNA synthesis, influencing whether transcription continues or stops.

## Contribution

The study identifies a regulatory helix that fine-tunes gene expression rather than acting as a simple ON–OFF switch for the 2′-deoxyguanosine-sensing riboswitch.

## Key findings

- The dGsw-fl riboswitch remains in an OFF state regardless of 2′-dG presence.
- The dGsw-int intermediate adopts an ON state with a stable anti-terminator helix in the absence of 2′-dG.
- 2′-dG binding inhibits anti-terminator helix formation, locking dGsw in an OFF state during RNA synthesis.

## Abstract

Transcriptional riboswitches, noncoding mRNA elements that operate in cis to regulate gene expression, have promising potential in medicine, synthetic biology, and directed evolution. They bind to cellular metabolites or metal ions with high specificity, leading to conformational rearrangements that facilitate the activation or premature termination of transcription for downstream genes. This elegant mechanism for feedback regulation of metabolic pathways has been identified in prokaryotes and a few eukaryotes. Our chemical probing of the 2′-deoxyguanosine (2′-dG)-sensing riboswitch demonstrates that the overall conformational state of the full-length riboswitch (dGsw-fl) is unresponsive to the 2′-dG. Although binding proceeds as expected, dGsw-fl exclusively populates an OFF state of transcriptional inhibition. We chemically probed the structure of a known dGsw transcriptional intermediate (dGsw-int) to evaluate the possibility of a cotranscriptional regulatory role. Interestingly, apo dGsw-int adopts an alternative conformation in which a stable anti-terminator helix is formed, leading to an ON state where transcription can proceed. In the presence of 2′-dG, this anti-terminator helix is destabilized to produce a conformation reminiscent of the full-length, OFF-state dGsw. Using a fluorescence quenching assay, we demonstrate that binding 2′-dG to early transcriptional intermediates can inhibit the formation of the anti-terminator helix, locking dGsw in an OFF state. These data suggest that metabolite sensing occurs during a brief window of time between the synthesis of two transcriptional intermediates. Our studies indicate that dGsw does not function as a binary ON−OFF switch but instead fine-tunes the transcription of downstream genes during RNA synthesis using key intermediates.