Isosteric and Nonisosteric Base Pairs in RNA Motifs: Molecular Dynamics and Bioinformatics Study of the Sarcin–Ricin Internal Loop

Abstract

The Sarcin-Ricin RNA motif (SR motif) is one of the most prominent recurrent RNA building blocks that occurs in many different RNA contexts and folds autonomously, i.e., in a context-independent manner. In this study, we combined bioinformatics analysis with explicit-solvent molecular dynamics (MD) simulations to better understand the relation between the RNA sequence and the evolutionary patterns of SR motif. SHAPE probing experiment was also performed to confirm fidelity of MD simulations. We identified 57 instances of the SR motif in a non-redundant subset of the RNA X-ray structure database and analyzed their basepairing, base-phosphate, and backbone-backbone interactions. We extracted sequences aligned to these instances from large ribosomal RNA alignments to determine frequency of occurrence for different sequence variants. We then used a simple scoring scheme based on isostericity to suggest 10 sequence variants with highly variable expected degree of compatibility with the SR motif 3D structure. We carried out MD simulations of SR motifs with these base substitutions. Non isosteric base substitutions led to unstable structures, but so did isosteric substitutions which were unable to make key base-phosphate interactions. MD technique explains why some potentially isosteric SR motifs are not realized during evolution. We also found that inability to form stable cWW geometry is an important factor in case of the first base pair of the flexible region of the SR motif. Comparison of structural, bioinformatics, SHAPE probing and MD simulation data reveals that explicit solvent MD simulations neatly reflect viability of different sequence variants of the SR motif. Thus, MD simulations can efficiently complement bioinformatics tools in studies of conservation patterns of RNA motifs and provide atomistic insight into the role of their different signature interactions.

How to understand atomistic molecular dynamics simulations of RNA and protein–RNA complexes?

“…Further, similar to experiments, different simulation tasks have different accuracy requirements. For example, when investigating structural dynamics of sarcin‐ricin loop (SRL), we wish to satisfy all its signature interactions . When complementing sequence alignments on substitutions in the SRL, we aim to achieve a qualitative ranking of the diverse substitutions which surpasses rankings by bioinformatics tools .…”

“…For example, when investigating structural dynamics of sarcin‐ricin loop (SRL), we wish to satisfy all its signature interactions . When complementing sequence alignments on substitutions in the SRL, we aim to achieve a qualitative ranking of the diverse substitutions which surpasses rankings by bioinformatics tools . In contrast, when simulating the whole ribosome, we do not expect structural details on a level of single non‐Watson‐Crick base pairs …”

New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch

“…S-turn motifs in other RNAs often participate in binding of proteins and RNA (12,(23)(24)(25) and are important for ribosome assembly, translation, and viral replication and packaging (12,19,(26)(27)(28). S-turn formation depends not only on base-base interactions but also on base-phosphate backbone interactions (29). The G37U and A57C single mutations reduced cross-linking, as predicted, but the G37U, A57C double substitution, which was predicted to introduce an isosteric pair at the 37-57 position, did not restore cross-linking efficiency (Fig.…”

RNABP COGEST: a resource for investigating functional RNAs

“…For example, a recent study on Sarcin-Ricin internal loop motif (SR-motif) reports two structurally observed variants of the reference native SR-motif (variant 0), characterized by isosteric substitutions of two different A:G H:S Trans base pairs, respectively. Variant 1 involves substitution of the A18:G4 H:S Trans base pair by C18:C4 H:S Trans, and variant 6 involves substitution of the A8:G15 H:S Trans base pair by A8:A15 H:S Trans ( 53 ) (see Supporting information S1 for details). In course of MD simulation, the C18:C4 base pair, in the flexible region of variant 1, showed far greater geometric fluctuations compared with the corresponding A18:G4 base pair in the variant 0.…”