Secondary Structure Predictions and Determination of Folding Pathways for TPP Riboswitch

Abstract

Riboswitches are regulatory segments in untranslated regions of mRNA that bind small molecules, resulting in a change in production of proteins encoded by the mRNA. The thiamine pyrophosphate (TPP) riboswitch modifies downstream gene expression based on the local concentration of TPP in multiple prokaryotes and eukaryotes. The mechanism of the riboswitch is mediated by its ability to form a secondary substructure known as aptamer that can adopt multiple three-dimensional configurations as a function of ligand binding. Strong experimental evidences that a magnesium ion plays an important role in the TPP-riboswitch interaction, altering the folding and binding energy landscape of the TPP riboswitch. However, the role played by the Mg2+ in stabilizing the confirmation of the riboswitch in its closing pathway remains unknown. Here, we report on the ligand binding mechanism of the eukaryotic Arabidopsis thaliana TPP riboswitch that is located upstream of 3’ untranslated intron region of thiC gene. We quantified the distinct opening and closing transitions of riboswitch structure at various TPP and Mg2+ concentrations at the single-molecule level with force spectroscopy, using an optical trap, and Forster resonance energy transfer (FRET). Our results suggest a model in which the closing mechanism of the riboswitch arms is dependent on Mg2+ concentration and undergoes multiple intermediate states that trap TPP into them to assume a stable off state.