Short talk 4
Resolving the conformational landscape of a large ribozyme by multi-trajectory smFRET

Presenting Author: Matteo Lisibach

Universität Zürich, Department of Chemistry, Winterthurerstrasse 190, 8057 Zürich, CH, susann.paulus@chem.uzh.ch

Author(s)
Matteo Lisibach¹, Abdul Rahman Sadiq¹, Prof. Roland K.O. Sigel¹, Dr. Susann Zelger-Paulus¹

¹ Universität Zürich

Single-molecule FRET (smFRET) is widely used to study RNA dynamics and is often applied using a single distance constraint. For large, multidomain RNAs such as our model system, this is not sufficient, as their underlying structural rearrangements are inherently complex. Here, we resolve the conformational landscape of a catalytic group II intron ribozyme undergoing multiple structural transitions during self-cleavage by integrating multiple FRET trajectories.

We introduced four labeling positions, yielding six pairwise FRET trajectories recorded sequentially, each reporting on distinct interdomain distances. While each dataset revealed a complex conformational landscape, correlating states across labeling schemes remained a major challenge. To overcome this, we disrupted a key tertiary interaction, reducing activity and redistributing conformational populations. This perturbation enabled mapping of corresponding states across all datasets. By integrating the trajectories, we identified three major conformational states and defined their domain organization.

Our results show that the ribozyme does not simply adopt a compact active structure but instead coordinates its domain arrangements in a functional manner. This work demonstrates that multi-trajectory smFRET, combined with targeted perturbations, can resolve the dynamic landscapes of large RNAs.

 

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