Conformational heterogeneity in the dGsw purine riboswitch: role of Mg²⁺ and 2’-dG in aptamer folding

Recent advancements in RNA structural biology have focused on unraveling the complexities of noncoding mRNA like riboswitches. These <i>cis</i>-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 prokaryotic genes involved in purine degradation and biosynthesis. They feature an aptamer domain organized around a three-way helical junction, where ligand encapsulation occurs at the junctional core. We chemically probed the aptamer domain of the 2'-dG-sensing purine riboswitch from <i>Mesoplasma florum</i> (dGsw) under various solution conditions to understand how Mg²⁺ and 2'-dG influence riboswitch folding. We find that 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 led to intramolecular competition between the alternate helix, P_alt, and P1. Mutational analysis confirmed that the 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 facilitate the formation of an antiterminator helix by opening the three-way junction surrounding the 2'-dG binding site. Our study establishes the importance of a closed terminal P1 helix conformation for metabolite binding and suggests that the delicate interplay between P1 and P_alt fine-tunes 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.