Characterization of Structure and Dynamics of the Guanidine-II Riboswitch from Escherichia coli by NMR Spectroscopy and Small-Angle X-ray Scattering (SAXS)

Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg2+ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg2+ and ligand binding.

Automated summary

Riboswitches are regulatory RNA elements that undergo conformational changes upon binding specific ligands. The guanidine-II riboswitch investigated here binds guanidinium and forms a kissing loop-loop interaction. Structural changes were studied using NMR spectroscopy, confirming the observed structure and characterizing the interaction with Mg2+ and ligand. Mutant constructs were also investigated to understand functional differences between the two hairpins P1 and P2. Formation of oligomeric states of the riboswitch induced by Mg2+ and ligand binding were observed through small-angle X-ray scattering (SAXS) and NMR DOSY data.