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Recent advancements in RNA structural biology have focused on unraveling the complexities of non-coding mRNA elements like riboswitches. These cis-acting regulatory regions undergo structural changes in response to specific cellular metabolites, leading to up or downregulation of downstream genes. The purine riboswitch family regulates many prokaryotic genes involved in purine degradation and biosynthesis. They feature an aptamer domain organized around a 3-way helical junction, where ligand encapsulation occurs at the junctional core. In our study, we chemically probed the aptamer domain of the 2’-dG-sensing purine riboswitch from Mesoplasma florum (dGsw) under various solution conditions to understand how Mg²⁺ and 2’-dG influence riboswitch folding. Here, we find that efficient 2’-dG binding strongly depends on Mg²⁺, indicating that Mg²⁺ is essential for priming dGsw for ligand interactions. We identified a previously undescribed sequence in the 5’ tail of dGsw that is complementary to a conserved helix. The inclusion of this region in a construct led to intramolecular competition between the alternate helix, Palt, and P1. Mutational analysis confirmed that 5’ flanking end of the aptamer domain forms an alternate helix in the absence of ligand. Molecular dynamics simulations revealed that this alternative conformation is stable. This helix may, therefore, facilitate the formation of an anti-terminator helix by opening the 3-way junction surrounding the 2’-dG binding site. Our study further establishes the importance of a closed terminal P1 helix conformation for metabolite binding and suggests that the delicate interplay between P1 and Palt may fine-tune downstream gene regulation. These insights offer a new perspective on riboswitch structure and enhance our understanding of the role that a conformational ensemble plays in riboswitch activity and regulation. 

Residing in the 5′ untranslated region of the mRNA, the 2′-deoxyguanosine (2′-dG) riboswitch mRNA element adopts an alternative structure upon binding of the 2′-dG molecule, which terminates transcription. RNA conformations are generally strongly affected by positively charged metal ions (especially Mg²⁺). We have quantitatively explored the combined effect of ligand (2′-dG) and Mg²⁺binding on the energy landscape of the aptamer domain of the 2′-dG riboswitch with both explicit solvent all-atom molecular dynamics simulations (99 μsec aggregate sampling for the study) and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) experiments. We show that both ligand and Mg²⁺are required for the stabilization of the aptamer domain; however, the two factors act with different modalities. The addition of Mg²⁺remodels the energy landscape and reduces its frustration by the formation of additional contacts. In contrast, the binding of 2′-dG eliminates the metastable states by nucleating a compact core for the aptamer domain. Mg²⁺ions and ligand binding are required to stabilize the least stable helix, P1 (which needs to unfold to activate the transcription platform), and the riboswitch core formed by the backbone of the P2 and P3 helices. Mg²⁺and ligand also facilitate a more compact structure in the three-way junction region. 

In the Big Data era, a change of paradigm in the use of molecular dynamics is required. Trajectories should be stored under FAIR (findable, accessible, interoperable and reusable) requirements to favor its reuse by the community under an open science paradigm.