Ligand Induced Folding Dynamics and Co-transcriptional Effect of TPP Riboswitch

Abstract

Non-coding RNA (ncRNA), which was initially considered as junk RNA, is increasingly being recognized as a common tool to regulate gene expression. Almost all stages of gene expression such as initiation, termination and maturation in both transcription and translation can be regulated by ncRNA. Unlike messenger RNA (mRNA) which plays a role as a code for the corresponding protein, ncRNA works not only as a functional sequence but also as a folding structure. These folding structures of ncRNA can have various functions like proteins, for example, binding a small ligand molecule, catalyzing cleavage reaction and associating with proteins. Riboswitches are typical examples of ncRNA and regulate gene expression by coupling ligand binding to a structural transition of the riboswitch, although the exact coupling mechanism remains unclear. We addressed this issue by characterizing both the ligand-free state of the Escherichia coli TPP (thiamine pyrophosphate) riboswitch aptamer and its structural transition upon ligand binding using single-molecule FRET (fluorescence resonance energy transfer). Our results reveal that the TPP aptamer dynamically samples a partially closed form resembling the holo-aptamer in the absence of a ligand but that TPP binding is not selective for the partially closed form, which is an unexpected finding in the light of the recently suggested conformational selection model. Additionally, we found that the preformation of the aptamer secondary structure is required for TPP binding, suggesting that TPP regulates gene expression by cotranscriptionally controlling the folding pathway of the TPP riboswitch. Recently, the fact that RNA folds sequentially as it is being transcribed is carefully considered for RNA structure prediction and dynamics study. It has been shown that this co-transcriptional effect strongly influences RNA folding pathway suggesting that the formation of transient conformations serves as a guideline for the following co-transcriptional folding. Elongation complex (EC) which works as a basic unit to produce RNA from DNA was successfully reconstituted for the co-transcriptional observation. By using a single-molecule FRET microscopy, we observed RNA folding process for individual molecules. We found that TPP riboswitch regulates the gene expression co-transcriptionally that means TPP concentration can influence the folding process during transcription. We expect that the EC reconstitution method for smFRET will be useful for structural studies of ncRNA.