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

Abstract

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.

Combining Coarse-Grained Simulations and Single Molecule Analysis Reveals a Three-State Folding Model of the Guanidine-II Riboswitch

“…It is possible that these high concentrations of the cationic molecule Gdm + are sufficient to emulate the effects of 5 mM divalent Mg 2+ . However, this might not be the case at higher RNA and/or lower ligand concentrations ( Schamber et al, 2021 ).…”

“…This also translates into assumptions that go into the treatment of dye linkers for FPS ( Kalinin et al, 2012 ; Dimura et al, 2016 ) or coarse-grained simulations. Finally, the concentrations used in single-molecule experiments may not be compatible with structural biology approaches or simulations ( Schamber et al, 2021 ), as can be seen in the experiments described above.…”

“…This interaction supposedly sequesters the anti-SD sequence, exposing the SD sequence and thus facilitating translation of the downstream gene ( Figure 1A ). Mg 2+ binding sites have been identified in each of the two stems ( Huang et al, 2017 ; Schamber et al, 2021 ). Crystallization could however only show homo-dimerization of isolated hairpins.…”

“…Using electron paramagnetic resonance (EPR) it was shown that RNAs comprising either hairpins P1 or P2 could form homo-as well as heterodimers ( Wuebben et al, 2020 ). Recently, a study combining nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS) showed interstrand kissing loop formation in an in vitro environment ( Schamber et al, 2021 ).…”

NMR of RNA - Structure and interactions

“…Recently, Mg 2þ requirements and guanidinium binding site were explored for guanidine II riboswitch with the use of NMR and paramagnetic relaxation enhancements, while diffusion-ordered NMR was used to discern between the intramolecular kissing loop and dimer formation. An ACGR (R is any nucleotide) loop was identified as vital for dimerization and guanidine binding [19]. The structure of ZMP (5-aminoimidazole-4-carboxamide ribonucleotide) sensing riboswitch and its ability to bind ligand was found to be affected by transcript length, enabling the sensing of conditions in the bacteria to regulate gene expression [20].…”