Site-Directed Spin Labeling Evidence of the Leader-Linker Interaction in the Glycine Riboswitch using Electron Paramagnetic Resonance Spectroscopy

Abstract

Riboswitches are mRNA transcripts that function to modulate genetic expression through selective recognition and binding of cognate ligands which, subsequently, induces RNA conformational changes. Continuous wave electron paramagnetic resonance (CW EPR) spectroscopy, when employed with site-directed spin labeling (SDSL), is a useful technique for investigating changes in site-specific dynamics within biological systems. In this work, SDSL CW EPR was used to study the leader-linker interaction in the Vibrio cholerae glycine riboswitch. The glycine riboswitch binds two molecules of glycine to regulate the expression of genes associated with glycine metabolism. The recently described leader-linker interaction in the glycine riboswitch has been investigated using biochemical methods and was shown to play a functional role in the ligand binding process. To probe local RNA backbone dynamics of select sites within the leader-linker interaction SDSL, using the R5 spin label, was employed. Incorporation of spin labels was achieved through the use of optimized ligation methodologies that allow small, synthetically modified RNA to be joined to the larger riboswitch RNA sequence. Empirical analysis of X-band EPR line shapes was used to characterize dynamics of the interaction at varying temperatures for differing folded states of the riboswitch in the absence or presence of salts and glycine ligand. Spectral variation at the labeled sites is in agreement with postulated secondary structural elements and has provided spectroscopic evidence in support of the leader-linker interaction.