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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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Magnesium ions mitigate metastable states in the regulatory landscape of mRNA elements
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.
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- PAR ID:
- 10597073
- Publisher / Repository:
- Cold Spring Harbor Laboratory Press
- Date Published:
- Journal Name:
- RNA
- Volume:
- 30
- Issue:
- 8
- ISSN:
- 1355-8382
- Page Range / eLocation ID:
- 992 to 1010
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Thiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on the Arabidopsis thaliana TPP riboswitch suggests a multistep TPP binding process involving multiple riboswitch configurational ensembles and Mg 2+ dependence underlies the mechanism of TPP recognition and subsequent transition to the expression-inhibiting state of the aptamer domain followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg 2+ in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain's sensor helices associated with TPP and Mg 2+ ligand binding occurred between at least five different ensembles on timescales ranging from µs to ms. These dynamics are orders of magnitude faster than the 10 sec-timescale folding kinetics associated with expression-state switching in the switch helix. Together, our results show that a TPP and Mg 2+ dependent mechanism determines dynamic configurational state ensemble switching of the aptamer domain's sensor helices that regulate the switch helix's stability, which ultimately may lead to the expression-inhibiting state of the riboswitch. Additionally, we propose that two pathways exist for ligand recognition and that this mechanism underlies a kinetic rheostat-like behavior of the Arabidopsis thaliana TPP riboswitch.more » « less
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Abstract Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are compounded for small RNAs as cryo-EM is inherently more difficult for lower molecular weight macromolecules. Here we present a strategy for fusing small RNAs to a group II intron that yields high resolution structures of the appended RNA. We demonstrate this technology by determining the structures of the 86-nucleotide (nt) thiamine pyrophosphate (TPP) riboswitch aptamer domain and the recently described 210-ntraiAbacterial non-coding RNA involved in sporulation and biofilm formation. In the case of the TPP riboswitch aptamer domain, the scaffolding approach allowed visualization of the riboswitch ligand binding pocket at 2.5 Å resolution. We also determined the structure of the ligand-free apo state and observe that the aptamer domain of the riboswitch adopts an open Y-shaped conformation in the absence of ligand. Using this scaffold approach, we determined the structure ofraiAat 2.5 Å in the core. Our versatile scaffolding strategy enables efficient RNA structure determination for a broad range of small to moderate-sized RNAs, which were previously intractable for high-resolution cryo-EM studies.more » « less
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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.more » « less
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SreA is one of seven candidate S-adenosyl methionine (SAM) class I riboswitches identified in Listeria monocytogenes, a saprophyte and opportunistic foodborne pathogen. SreA precedes genes encoding a methionine ATP-binding cassette (ABC) transporter, which imports methionine, and is presumed to regulate transcription of its downstream genes in a SAM-dependent manner. The proposed role of SreA in controlling the transcription of genes encoding an ABC transporter complex may have important implications for how the bacteria senses and responds to the availability of the metabolite SAM in the diverse environments in which L. monocytogenes persists. Here we validate SreA as a functional SAM-I riboswitch through ligand binding studies, structure characterization, and transcription termination assays. We determined that SreA has both a similar structure and SAM binding properties to other well characterized SAM-I riboswitches. Despite apparent structural similarities to previously described SAM-I riboswitches, SreA induces transcription termination in response to comparatively lower (nM) ligand concentrations. Furthermore, SreA is a leaky riboswitch that permits some transcription of the downstream even in the presence of mM SAM suggesting that L. monocytogenes may “dampen” the expression of genes for methionine import, but likely does not turn them “OFF”.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1261/rna.079767.123
