Ligand-mediated and tertiary interactions cooperatively stabilize the P1 region in the guanine-sensing riboswitch

Abstract

Riboswitches are genetic regulatory elements that control gene expression depending on ligand binding. The guanine-sensing riboswitch (Gsw) binds ligands at a three-way junction formed by paired regions P1, P2, and P3. Loops L2 and L3 cap the P2 and P3 helices and form tertiary interactions. Part of P1 belongs to the switching sequence dictating the fate of the mRNA. Previous studies revealed an intricate relationship between ligand binding and presence of the tertiary interactions, and between ligand binding and influence on the P1 region. However, no information is available on the interplay among these three main regions in Gsw. Here we show that stabilization of the L2-L3 region by tertiary interactions, and the ligand binding site by ligand binding, cooperatively influences the structural stability of terminal base pairs in the P1 region in the presence of Mg2+ ions. The results are based on molecular dynamics simulations with an aggregate simulation time of ~10 μs across multiple systems of the unbound state of the Gsw aptamer and a G37A/C61U mutant, and rigidity analyses. The results could explain why the three-way junction is a central structural element also in other riboswitches and how the cooperative effect could become contextual with respect to intracellular Mg2+ concentration. The results suggest that the transmission of allosteric information to P1 can be entropy-dominated.

Unraveling RNA dynamical behavior of TPP riboswitches: a comparison between Escherichia coli and Arabidopsis thaliana

“…Indeed, both average RMSD values per region and the overall pattern of fluctuations were similar for all systems. These values are consistent with RMSD values seen in other studies reporting simulations of small RNA aptamers 21 – 24 which ranged between 3–4 Å approximately.…”

Tertiary Interactions in the Unbound Guanine-Sensing Riboswitch Focus Functional Conformational Variability on the Binding Site

“…The simulation systems for the REMD simulations were prepared following the protocol used earlier. , In detail, we set up two variants of the guanine-sensing riboswitch aptamer domains (Gsw): The wild-type system, also referred to as Gsw apt , and a G37A/C61U mutant, referred to as Gsw loop . The RMSD between both starting structures is ∼0.8 Å (Figure S2 in the Supporting Information).…”

“…For both variants, two simulation systems were set up as described earlier, using different Mg 2+ concentrations (0 and 20 Mg 2+ ions per RNA molecule, respectively) based on experimental findings on the Mg 2+ dependence of Gsw loop stability. , Following our earlier findings on the importance of initial Mg 2+ ion placement, Mg 2+ ions were added with a first hydration shell to allow for initial mobility of the ions, using the protocol described earlier . In particular, such hexahydrate Mg 2+ ions were placed at locations of the Gsw structures where Co­(NH 3 ) 6 ions were found in the crystal structures. ,, Subsequently, the system was placed in an octahedral box of TIP3P water molecules, such that the distance between the edges of the boxes and the closest solute molecule was at least 11 Å. Na + ions were added as counterions in order to neutralize the net charge of the simulation system, yielding a final system size of ∼50 000 atoms.…”

“…The thermalization of the generated systems was done based on a protocol applied earlier. ,, First, the generated system was minimized by 200 steps of steepest descent minimization, followed by 50 steps of conjugate gradient minimization. Subsequently, the system was initially heated from 100 K to 300 K performing canonical ensemble (NVT)-MD simulations for 50 ps.…”

Pseudo aptamer expands aptamer’s applications

“…The effect often attributes to both non-specific and specific interactions, since the recognition between nucleotide acid and proteins involves side chains and backbone interactions in the manner of the nucleic acid bases amino acid codes, 47,48 hydrogen bonds, [49][50][51][52][53] charge-charge interactions, [54][55][56][57][58][59] hydrophobicity or hydrophilicity 60,61 conformations, 62-69 rigidity etc. [70][71][72] Among the modifications on backbone, sulfurization is one of common ways. In doing so, dithiazol-one based derivatives as the most efficient sulfurization reagents have been employed to introduce sulfur element.…”