Cotranscriptional folding kinetics of Cbe pfl riboswitch

The function of the riboswitch is determined by the rearrangement behavior of intermediate structures that emerge during cotranscriptional folding. These folded fragments interact with ligands, alter their structure and thereby regulate gene expression. We conducted theoretical investigations into the cotranscriptional folding behavior of the Clostridium beijerinckii pfl riboswitch, both in the absence and presence of the ligand, using a computational helix-based method. To simplify conformations, we employed the Conformational Resampling through the Kinetic Relaxation (CRKR) method. We varied factors like the ligand concentration, the transcription rate, and the transcription pausing sites. Notably, we identified two different folding paths of Cbe pfl riboswitch depending on whether the ligand exists or not. It has also been investigated that the higher ligand concentration and the higher transcription rate can both enhance the formation of antiterminated structure. During the cotranscriptional pausing event, pausing at C107 and C110 can reduce the final population of the antiterminated structures while pausing at the site U85 will stabilize the formation of an important intermediate transition structure N8 The obtained results are in excellent agreement with the experimental data, and these investigations provide novel insights into the transition of conformational space during the cotranscriptional folding process of RNAs . It demonstrates the folding process of Cbe pfl riboswitch when ligand is present. The pathway structure of N1 N6 is the same as that the ligand i s not bound. New N7 (89 steps) and N8 (114 steps) structures are generated when the ligand binds to the aptamer region. ∆G N7 = −23.35 kcal/mol and ∆G N8 = −30.79 kcal/mol are the free energies of N7 and N8. The final stucture is antiterminator. • Two main terminator/antiterminator action pathways with and without ligand binding are identified. • Higher ligand concentration and transcription rate enhance antiterminated structure formation when the ligand is present. • Pausing at C107 and C110 can reduce the final population of antiterminated structure while pausing at U85 can not. • The population of intermediates during cotranscriptional folding largely depend on the cotranscription rate.