An intricate balance of hydrogen bonding, ion atmosphere and dynamics facilitates a seamless uracil to cytosine substitution in the U-turn of the neomycin-sensing riboswitch

Abstract

The neomycin sensing riboswitch is the smallest biologically functional RNA riboswitch, forming a hairpin capped with a U-turn loop—a well-known RNA motif containing a conserved uracil. It was shown previously that a U→C substitution of the eponymous conserved uracil does not alter the riboswitch structure due to C protonation at N3. Furthermore, cytosine is evolutionary permitted to replace uracil in other U-turns. Here, we use molecular dynamics simulations to study the molecular basis of this substitution in the neomycin sensing riboswitch and show that a structure-stabilizing monovalent cation-binding site in the wild-type RNA is the main reason for its negligible structural effect. We then use NMR spectroscopy to confirm the existence of this cation-binding site and to demonstrate its effects on RNA stability. Lastly, using quantum chemical calculations, we show that the cation-binding site is altering the electronic environment of the wild-type U-turn so that it is more similar to the cytosine mutant. The study reveals an amazingly complex and delicate interplay between various energy contributions shaping up the 3D structure and evolution of nucleic acids.

Molecular mechanisms for dynamic regulation of N1 riboswitch by aminoglycosides

“…In the absence of aminoglycosides, the WC-edge hydrogen bonds between U10 and U21 often shift from conformation I to conformation II as shown in Figure 4B . Similar observations on the conformations of the U10–U21 pair were also made in recent simulations of the N1 riboswitch with and without RIO ( 48 ).…”

Improving The Performance Of The Amber Rna Force Field By Tuning The Hydrogen-Bonding Interactions

“…Note that all analyses involving occurrence of the native state within generated conformational ensembles considered just four signature H-bonds for the UNCG TL, i.e., we counted only the GL4(N1H)…UL1(O2) interaction from the bifurcated GL4(N1H/N2H)…UL1(O2) H-bond. three-tetrad DNA quadruplex stems 65 while the modified phosphate parameters were not successful in correcting base-phosphate H-bonding in simulations of Neomycin-sensing riboswitch, 66 indicating that the modified phosphate parameters with OPC water model do not represent the ultimate solution for tuning the phosphate-group interactions. The second problem was destabilization of the native folded state by underestimation of the native H-bonds including the stem base pairing.…”

Phosphorothioate Substitutions in RNA Structure Studied by Molecular Dynamics Simulations, QM/MM Calculations and NMR Experiments

“…Parameters for the ribostamycin ligand and for protonated cytosine C14 + were taken from earlier simulation study of the NSR. 27 Each system was processed by a series of minimizations and equilibrations as described in the Supporting information. The reference wild-type and mutC14 simulations with no chemical modifications had simulation times of 10 microseconds, while the thiomodified substrates were simulated in four parallel simulations, each one-microsecond long (SI Appendix Table S1).…”