Comparative Study between Transcriptionally- and Translationally-Acting Adenine Riboswitches Reveals Key Differences in Riboswitch Regulatory Mechanisms

Abstract

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.

Author Summary

Bacterial genetic regulation is mostly performed at the levels of transcription and translation. Recently discovered riboswitches are RNA molecules located in untranslated regions of messenger RNAs that modulate the expression of genes involved in the transport and metabolism of small metabolites. Several riboswitches have recently been shown to employ various regulation mechanisms, but no general rules have yet been deduced from these studies. Here, we have analyzed two adenine-sensing riboswitches of Bacillus subtilis and Vibrio vulnificus that differ by the level at which they control gene expression, which is transcription and translation, respectively. We find that, beyond the obvious difference in transcriptional and translational modulation, riboswitch regulation mechanisms of both adenine riboswitches are fundamentally different. For instance, while the adenine riboswitch from B. subtilis performs co-transcriptional binding for gene regulation, the riboswitch from V. vulnificus relies on reversible ligand binding to achieve gene regulation during mRNA translation. In agreement with co-transcriptional binding of the B. subtilis riboswitch, we find that transcriptional pausing is crucial for gene 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.