Effect of Magnesium Ions on Thiamine Pyrophosphate\u27s (TPP) Ability to Stabilize the Closed State of the TPP Riboswitch

Riboswitches are genetic switches in messenger RNA that regulate gene expression through mechanisms. These include ribosome binding site sequestration and premature transcription termination, in response to the presence of metabolites such as thiamine pyrophosphate (TPP). There is strong experimental evidence for a distinct energy landscape as the TPP Riboswitch folds and binds the TPP, and that magnesium plays an important role in the TPP-riboswitch interaction. However, the quantitative role played by the magnesium ion in stabilizing the riboswitch’s closing pathway confirmation remains unknown. To quantify the role of magnesium in the TPP riboswitch operation, we performed single-molecule optical tweezer experiments to measure the folding and unfolding energy landscape in the presence of varying concentrations of magnesium. We made heterobifunctional DNA handles using PCR amplification that we will attach to the 5\u27 and 3\u27 ends the riboswitch. We ligated the handles with a linking DNA oligo and gel purified the product. By tethering one end of the ligated DNA handle to the coverslip and the other to a 1 μm diameter bead in the optical trap, we measured the stretching force-displacement curves of the DNA handles in the optical tweezer and found that the beads were becoming attached to multiple strands of DNA at once. In the future, we will be able to understand the effects of magnesium concentration on the riboswitch binding and following pathways, as well as the role of the TPP Riboswitch in gene regulation