Speaker
Description
RNA polymerase III (Pol III) synthesizes short RNAs with exceptional efficiency. Unlike Pol II, Pol III retains TFIIE- and TFIIF-like sub-complexes that give it unique capabilities during different stages in transcription. Yet the role of the tandem-winged-helix (tWH) domain of Rpc34 during elongation has remained unresolved, in part because its density is consistently missing from cryo-EM structures of Pol III elongation complexes (ECs). Here, we used single-molecule Förster resonance energy transfer (smFRET) to determine the dynamics and position of Rpc34 WH2 within quiescent Pol III ECs. To enable site-specific dye labeling, we incorporated an azide-bearing unnatural amino acid compatible with strain-promoted click chemistry and implemented a simple thiol-capping strategy to suppress previously overlooked alkyne-thiol cross-reactivity. With the acceptor at Rpc34 WH2 and the donor at a defined position on the DNA template as the reference, we observed three discrete FRET states. A substantial fraction of Pol III ECs dynamically transitioned among these states with characteristic kinetics, indicating that Rpc34 WH2 engages the EC through transient, weak and promiscuous interactions. Nano-positioning triangulation revealed three corresponding Rpc34 WH2 docking sites across the Pol III DNA-binding cleft－ downstream, middle and upstream－including a position that matches its pre-initiation complex (PIC) location. Together with prior Pol I and Pol II studies, these findings clarify previously elusive roles of Rpc34 WH2 in elongation, particularly its contribution to stabilizing Pol III EC. Additionally, the bio-orthogonal labeling framework developed here provides a generalizable route for applying smFRET to large native protein assemblies.
