Dynamic positioning of a TFIIE-related winged-helix domain in RNA polymerase elongation complex III underpins its multi-functionality
Wu, J.-S.; Lin, Y.-C.; Wei, Y.-Y.; Lin, H.-H.; Liu, Y.-C.; Chang, J.-W.; Tu, I.-P.; Chen, H.-T.; Chang, W.-H.
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The tandem-winged-helix (tWH) domain of RNA polymerase III (Pol III) Rpc34 subunit is a multi-function hub in Pol III apparatus while the structure basis remains elusive. To probe tWH in Pol III elongation complex (EC), we engineered an azide-bearing unnatural amino acid into tWH and implemented a thiol-capping scheme to enable strain- promoted click reaction for selective dye labeling to suit single-molecule Forster resonance energy transfer (smFRET). Our discrete smFRET results and nano-positioning analysis reveal multiple docking sites of Rpc34 WH2 on Pol III EC with characteristic dwell-times, reflecting promiscuous and weak interactions underpinning tWHs multi-functionality. The docking sites include one overlapping with that during initiation and others on transcription bubble and downstream DNA, previously unreported. This work provides mechanistic insights into Pol III transcription re-initiation and elongation, with useful bio-orthogonal strategies for studying structural dynamics of large native protein complexes.
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