Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

56-POS Board 28 SITE-SPECIFIC FLUORESCENT LABELING ENABLES SINGLE-MOLECULE STUDIES OF ENDOGENOUS HUMAN RNA POLYMERASE I Andreas Schmidbauer 1,2 ; Julia L Daiß 2 ; Gerti Beliu 3,4 ; Christoph Engel 2 ; Dina Grohmann 1,2 ; 1 University of Regensburg, Institute of Microbiology & Archaea Centre, Single-Molecule Biochemistry Lab, Regensburg, Germany 2 University of Regensburg, Regensburg Center for Biochemistry, Regensburg, Germany 3 University of Regensburg, Departement for Chemistry and Pharmacy, Bioimaging, Regensburg, Germany 4 University of Würzburg, Rudolf Virchow Center for Integrative and Translational Bioimaging, Würzburg, Germany Ribosomal RNAs (rRNAs) constitute the structural and catalytic core of ribosomes, making RNA polymerase I (Pol I)-mediated rRNA synthesis essential for cellular homeostasis. A unique feature of Pol I is the stable incorporation of the auxiliary subunits A49 and A34 in addition to the core enzyme shared with Pol II and Pol III. Among these, A49 is essential for the transition from transcription initiation to elongation, ensuring efficient rRNA production. Single-molecule FRET (smFRET) studies of prokaryotic and lower eukaryotic transcription systems have provided key insights into transcription mechanisms and dynamics. However, comparable studies of Pol I are lacking because direct fluorescent labeling of large human protein complexes remains technically challenging. Here, we established a method for site-specific fluorescent labeling of human Pol I compatible with single-molecule studies. We combined genetic code expansion (GCE) with a CRISPR-Cas9-engineered HEK cell line expressing FLAG-sfGFP tagged Pol I. GCE enables incorporation of unnatural amino acids into individual Pol I subunits overexpressed in these cells. We demonstrate that the modified subunits are incorporated into endogenous Pol I complexes. Subsequent click chemistry allowed site-specifically coupling of fluorophores to the engineered positions. The resulting fluorescently labeled Pol I complexes retained catalytic activity. Using smFRET, we localized the position of A49 within a transcription elongation complex. In addition, intramolecular FRET measurements with a doubly labeled Pol I variant revealed conformational states of the A49 tandem winged-helix domain. Together, this work establishes a versatile platform for the site-specific fluorescent labeling of endogenous human Pol I complexes and enables direct monitoring of the essential Pol I-specific transcription factor A49. The approach overcomes a major technical barrier for fluorescence-based studies of human transcription and provides a foundation for future single molecule investigations on structure and dynamics of human multi-subunit proteins.

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