Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Wednesday Speaker Abstracts

DECIPHERING THE MECHANISMS OF MOLECULAR MACHINES THAT UNWIND AND UNTANGLE NUCLEIC ACIDS BY SINGLE-MOLECULE FRET: FROM RETROSPECTIVE TO PERSPECTIVE

Dagmar Klostermeier 1 ; 1 University of Münster, Münster, Germany

Topoisomerases catalyze the inter-conversion of DNA topoisomers, including the introduction and removal of supercoils, catenation and decatenation, and knotting and unknotting of DNA. Enzymes of the type IIA family, comprising eukaryotic topoisomerase II (Topo II) and the bacterial enzymes gyrase and topoisomerase IV (Topo IV), share a common architecture, but catalyze different reactions in vitro , and have different functions in vivo : Topo II catalyzes the relaxation and decatenation of DNA in vitro , and is responsible for the decatenation of DNA in vivo . Gyrase introduces negative supercoils into DNA and catalyzes DNA decatenation. In vivo , it is responsible for the removal of positive supercoils accumulating during transcription and replication. Topo IV can relax and decatenate DNA in vitro , and untangles precatenanes and catenanes arising during replication in vivo . All of these reactions are ATP-dependent. In the first part, I will present single-molecule FRET studies in which we have dissected conformational changes in the catalytic cycles of these enzymes in the different reactions they catalyze. We show that they populate different conformational intermediates, pointing to differences in the underlying mechanisms. We also show differences in the mechanisms of the same enzyme from different species, and in the mechanism of the same enzyme catalyzing different reactions. Topoisomerases are common targets in therapeutic intervention. Our results therefore have important implications for topoisomerase inhibition and drug development. In the second part, I will present single-molecule FRET studies that identified conformational changes in the catalytic cycle of RNA helicases of the DEAD-box family. These enzymes catalyze the ATP-dependent unwinding of RNA duplex elements. We show that they switch from an open to a closed state on the cooperative binding of ATP and RNA. This closing event leads to a local destabilization of the duplex and unwinding. Phosphate release after ATP hydrolysis resets the enzyme to the open state for further catalytic cycles. Using distance restraints from single-molecule FRET, we have identified the position of an RNA-binding domain flanking the helicase core of a bacterial helicase, and have shown that a substantial movement of this domain on RNA binding leads to allosteric activation of the helicase core. The translation factor eIF4A is a minimal DEAD-box helicase consisting just of the helicase core. We show that it can exist in three states: an open state in the absence of ligands, a half-open state stabilized by eIF4G, and a closed state when eIF4G, RNA and ATP are bound. From single molecule experiments on immobilized eIF4A, we derive rate constants for opening and closing of eIF4A and demonstrate that eIF4B and eIF4G modulate eIF4A activities through modulation of its conformational cycle. The length, sequence and structure of the RNA substrate also regulates eIF4A activity through conformational control. We present sequence-encoded imaging, a multiplexed single-molecule FRET approach coupled to next-generation sequencing, that enables us to study this effect of RNA on eIF4A dynamics on a transcriptome-wide scale.

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