Directional co-transcriptional folding and pausing create kinetic checkpoints for riboswitch-controlled gene...

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Regulation

Background:

• Transcriptional riboswitches control gene expression by binding small molecules.
• Directly observing ligand binding to riboswitches during transcription is challenging due to labeling limitations.

Purpose of the Study:

• To visualize and understand the co-transcriptional binding of tRNA ^Gly to the GlyQS T-box riboswitch.
• To elucidate the role of RNA folding pathways and transcriptional pausing in riboswitch-mediated gene regulation.

Main Methods:

• Single-molecule fluorescence microscopy to observe dynamic RNA-protein interactions.
• Investigating the influence of transcription rate and pausing on riboswitch-tRNA complex formation.

Main Results:

• Visualized dynamic sampling of riboswitch structural domains by tRNA ^Gly during transcription.
• Demonstrated that a 5'-to-3' hierarchical folding pathway, influenced by transcription rate and pausing, dictates productive ligand recognition.
• Showed that pausing stabilizes the riboswitch-tRNA complex and promotes selective RNA polymerase readthrough.

Conclusions:

• Established a kinetic checkpoint model where pausing converts transient tRNA binding into stable regulatory decisions.
• Identified directional co-transcriptional folding and transcriptional pausing as key mechanisms for robust and selective riboswitch function.