Single-molecule Ligand Response of Guanidine-IV riboswitch

Abstract

Riboswitches are non-coding RNA elements that can specifically sense ligands and regulate gene expression. A recent report introduced a type of riboswitch known as the guanidine-IV riboswitch, which responds to guanidine levels and controls the transcription of downstream genes. However, there is currently a lack of clarity regarding the mechanism by which the riboswitch senses guanidine and undergoes conformational changes. This lack of understanding has impeded the application of this riboswitch. Therefore, our study aimed to address this gap by investigating the conformational changes that occur in a guanidine-IV riboswitch RNA. We examined the aptamer, terminator, and full-length riboswitch using single-molecule fluorescence resonance energy transfer (smFRET). Our findings indicated that the aptamer portion was more sensitive to guanidine compared to the terminator and full-length riboswitch. Additionally, we mimicked the structural changes in the guanidine-IV riboswitch that occur during transcription at a single-nucleotide and single-molecule level using Position-specific Labelling of RNA (PLOR) and smFRET. The results showed that guanidine caused a less pronounced change in the riboswitch RNA after transcribing 88 nucleotides. Based on our findings, we have proposed a folding model for the guanidine-IV riboswitch in the absence and presence of guanidine.The guanidine-IV riboswitch’s aptamer domain exhibits a greater sensitivity towards guanidine in comparison to both the terminator and full-length riboswitch. This results in the riboswitch showcasing a narrow transcriptional window that is responsive to ligand binding. And a comprehensive structure-function model for the guanidine-IV riboswitch under both guanidine-free and guanidine-present conditions.