Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

27-POS Board 13 TWO IN ONE: METAL-ION DEPENDENT BINDING AND FOLDING OF AN RIBOSOMAL RNA TERTIARY CONTACT STUDIED BY MEANS OF ENSEMBLE AND SINGLE-MOLECULE FRET Mara Henschel 1 ; Mirko Weber 1 ; Vanessa Schumann 1 ; Michael Schlierf 2 ; Richard Börner 1 ; 1 Laserinstitut Hochschule Mittweida, Mittweida, Germany 2 Technische Universität Dresden, Dresden, Germany The orchestrated interplay of RNA tertiary contacts shapes the folding landscape during ribosomal maturation. Here, we investigate two complementary folding trajectories of a fluorescently labelled (Cy3/5) ribosomal RNA tertiary contact from Saccharomyces cerevisiae. These trajectories report on a kissing-loop (KL) formation acting as GAAA tetraloop (TL) receptor and a KL-TL tertiary-contact formation, both at the ensemble and the single-molecule level. The metal-ion-dependent folding was followed at room temperature during K(I) and Mg(II) titrations using ensemble and pulsed-interleaved excitation FRET (PIE-FRET) together with polarization resolved detection to assess dye-dye distances and mobility, potential dye– RNA interactions and RNA domain dynamics. smFRET histograms show the expected shift towards higher FRET efficiencies upon addition of metal ions, indicating progressive compaction during RNA folding and in agreement with ensemble FRET measurements. At low monovalent metal ion concentration smFRET reveals a broad conformational heterogeneity of possible conformations invisible in the ensemble regime. A pronounced monovalent metal-ion sensitivity for the kissing loop and the expected divalent metal-ion sensitivity for the GAAA tetraloop binding, i.e., the tertiary contact formation, was found as expected. Comparing poly-U and poly-A linkers in the RNA model system reveal slightly higher FRET efficiencies and broader distributions for poly-A linker which were attributed to pronounced stacking interactions in comparison to a more flexible poly-U linker. Burst variance analysis (BVA) was further used to characterize the conformational ensemble of the folded/unfolded state of the KL and the bound/unbound state of the TL, respectively. Finally, fully corrected FRET histograms were used for FRET-guided integrative modelling and yield an RNA 3D structure based mechanistic model of the metal-ion dependent folding trajectory of this rRNA tertiary contact.

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