Human RNA polymerase III termination favors decomposition over facilitated recycling.

Eukaryotic transcription termination remains incompletely understood. Using single-molecule fluorescence assays, we examined intrinsic termination by human RNA polymerase III (RNAP3) and identified mechanistic trifurcation leading to readthrough and two termination pathways with distinctive dissociation kinetics. The dominant pathway, decomposing termination, involves (near-)simultaneous dissociation of RNA and DNA from RNAP3, resulting in complex decomposition. In contrast, recycling termination, where RNA is released while RNAP3 remains DNA-bound and one-dimensionally diffuses to enable facilitated recycling, accounts for 2-14% of transcription, diverging from its predominance in bacterial intrinsic termination. Confirming that robust termination requires a nontemplate-strand T-tract of four or more consecutive thymidines, we uncover that longer T-tracts favor recycling over decomposing termination, reflecting mechanistic distinction between the two pathways. While upstream RNA hairpins enhance termination efficiency, their impact decreases with distance from T-tracts, affecting both pathways similarly. Kinetically, transcript release takes consistently longer in decomposing than recycling termination, aligning with polymerase displacement versus RNA shearing. Furthermore, an RPC10 subunit mutation causes recurrent elongation arrest with differential effects on termination pathways, revealing its selective role in governing this branching process. These findings reinforce evolutionary conservation of termination-pathway duality and highlight diversion of pathway preference in human RNAP3 termination, establishing its fundamental mechanistic principles.