Dynamic positioning of a TFIIE-related winged-helix domain in RNA polymerase elongation complex III underpins its multi-functionality

RNA polymerase III (Pol III) is a large protein complex that efficiently synthesizes structural and non-translated RNAs. The tandem-wing-helix (tWH) domain (WH1 and WH2) in the Rpc34 subunit of the TFIIE-related sub-complex plays multiple roles central to Pol III transcription, but the structural basis remains conundrum due to its cryo-EM density missing in enlongation complex (EC). Here, by site-specifically incorporating an azido unnatural amino acid (UAA) into Pol III Rpc34 WH2 and developing a simple protocol to enable selective labeling using strain-promoted azide-alkyne cycloadditio—the Bertozzi click reaction, we probed Rpc34 WH2 dynamics using single-molecule Förster resonance energy transfer (smFRET) between an acceptor in Rpc34 WH2 and a donor in DNA. Our smFRET resolved multiple FRET states with inter-state transitions exhibiting characteristic kinetics. Further nano-positioning analysis localized the docking positions of those FRET states on Pol III EC, including sites related to initiation and transcription bubble management. Our findings of Rpc34 WH2 dynamical positioning suggest the docking interactions are weak and promiscuous, providing a mechanism to rationalize multi-functionality of Pol III Rpc34 tWH. This study expands our understanding of Pol III transcription machinery and our chemical biology means are widely applicable to explore structural dynamics of large protein complexes.