Co-transcriptional folding as a key factor in riboswitch function

In the realm of gene regulation, understanding the precise mechanisms by which RNA molecules fold and function during transcription is crucial. This study delves into this intricate process, focusing on the type I-A 2′-deoxyguanosine (2'dG)-sensing riboswitch from Mesoplasma florum. By employing NMR spectroscopy and computational modeling, we have uncovered critical insights into how co-transcriptional folding influences ligand binding and conformational switching in these regulatory RNAs.

Riboswitches are small, metabolite-sensing RNA molecules that regulate gene expression by undergoing structural changes upon binding to specific ligands. These conformational switches can either terminate or promote transcription, depending on the presence of the ligand. Despite extensive studies on riboswitches, the relationship between the length of the mRNA transcript, ligand binding, and the subsequent allosteric conformational changes has remained elusive—until now.

Investigating co-transcriptional folding

The study focuses on the I-A type 2'dG-sensing riboswitch, exploring how it folds and functions during transcription. By mapping changes in the conformational space of transcription intermediates at single-nucleotide resolution, we provide a detailed picture of the folding process. This meticulous approach reveals a fine-tuning mechanism where ligand binding and sequence-dependent alterations of the RNA structure are closely linked to the transcript length.

Key findings

Using NMR spectroscopy, we analyzed transcription intermediates with single-nucleotide precision, offering unprecedented insights into the folding process of the riboswitch. The study highlights the importance of metastable states—temporary conformations that the RNA adopts during transcription. These states are crucial for the proper functioning of the riboswitch, ensuring that the RNA can efficiently switch structures in response to ligand binding. The findings underscore that RNA folding is highly sequence-dependent, with specific sequences promoting or hindering the formation of certain structures. This dependency plays a critical role in the riboswitch's ability to regulate gene expression.

Implications for synthetic biology and healthcare

Understanding the co-transcriptional folding of riboswitches opens up new avenues for the rational design of inducible molecular devices. These devices could be tailored for various applications in synthetic biology and healthcare, such as creating new biosensors or developing targeted gene regulation therapies. The knowledge gained from this study provides a framework for designing RNA molecules with precise regulatory functions, paving the way for innovative biotechnological solutions.


NMR Structural Profiling of Transcriptional Intermediates Reveals Riboswitch Regulation by Metastable RNA Conformations
Christina Helmling, Anna Wacker, Michael T. Wolfinger, Ivo L. Hofacker, Martin Hengsbach, Boris Fürtig, Harald Schwalbe
J. Am. Chem. Soc. 139 (7):2647–56 (2017) | doi:10.1021/jacs.6b10429

See also

Efficient Computation of Cotranscriptional RNA-Ligand Interaction Dynamics
Michael T. Wolfinger, Christoph Flamm, Ivo L. Hofacker
Methods 143:70–76 (2018) | doi:10.1016/j.ymeth.2018.04.036 | Preprint PDF