RNA folding kinetics control riboswitch sensitivity in vivo

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Riboswitches are crucial RNA elements regulating gene expression in response to small molecules.
• Understanding riboswitch sensitivity (EC50) is key to their diverse biological roles and potential applications.
• Conserved aptamer domains in riboswitches exhibit varying sensitivities across different cellular contexts.

Purpose of the Study:

• To investigate the role of RNA folding dynamics in controlling riboswitch sensitivity.
• To identify mechanisms by which RNA folding influences ligand responsiveness.
• To demonstrate the general applicability of these findings across different riboswitch types.

Main Methods:

• Investigated the Clostridium beijerinckii pfl ZTP riboswitch.
• Identified sequence and structural modifications to the expression platform that slow RNA folding.
• Applied these modifications to diverse riboswitches with varying architectures and regulatory mechanisms.

Main Results:

• Multiple mechanistic routes were found to slow RNA folding, consistently enhancing riboswitch sensitivity.
• Slowing expression platform folding sensitizes riboswitches operating in a kinetic regime.
• Findings were validated across riboswitches with diverse aptamer domains and regulatory strategies.

Conclusions:

• RNA folding dynamics are critical determinants of riboswitch sensitivity.
• Strategies to slow expression platform folding can be used to rationally program riboswitch sensitivity.
• These findings provide general principles for understanding and engineering dynamic RNA systems.