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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.
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Cotranscriptional RNA strand exchange underlies the gene regulation mechanism in a purine-sensing transcriptional riboswitch
Abstract RNA folds cotranscriptionally to traverse out-of-equilibrium intermediate structures that are important for RNA function in the context of gene regulation. To investigate this process, here we study the structure and function of the Bacillus subtilis yxjA purine riboswitch, a transcriptional riboswitch that downregulates a nucleoside transporter in response to binding guanine. Although the aptamer and expression platform domain sequences of the yxjA riboswitch do not completely overlap, we hypothesized that a strand exchange process triggers its structural switching in response to ligand binding. In vivo fluorescence assays, structural chemical probing data and experimentally informed secondary structure modeling suggest the presence of a nascent intermediate central helix. The formation of this central helix in the absence of ligand appears to compete with both the aptamer’s P1 helix and the expression platform’s transcriptional terminator. All-atom molecular dynamics simulations support the hypothesis that ligand binding stabilizes the aptamer P1 helix against central helix strand invasion, thus allowing the terminator to form. These results present a potential model mechanism to explain how ligand binding can induce downstream conformational changes by influencing local strand displacement processes of intermediate folds that could be at play in multiple riboswitch classes.
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- PAR ID:
- 10365665
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 50
- Issue:
- 21
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- p. 12001-12018
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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