What question did this study set out to answer?

This study aims to clarify the mechanisms of transcription termination by human RNA polymerase III, focusing on distinguishing between decomposition and recycling pathways.

May 25, 2026Open Access

Human RNA polymerase III termination favors decomposition over facilitated recycling

SHSun HanSeoul National University QWQianmin WangFudan University ESEunho SongNational Institutes of Health

Key Points

This study aims to clarify the mechanisms of transcription termination by human RNA polymerase III, focusing on distinguishing between decomposition and recycling pathways.
Utilized single-molecule fluorescence assays to examine intrinsic termination by RNAP3.
Identified two termination pathways with distinctive kinetics: decomposing and recycling termination.
Evaluated the effects of T-tract lengths and upstream RNA hairpins on termination efficiency.
The decomposing termination pathway accounts for the majority of transcription termination events, involving simultaneous dissociation of RNA and DNA.
Recycling termination represents only 2–14% of events, showing a marked difference from bacterial systems.
A mutation in the RPC10 subunit leads to elongation arrest and impacts the preference for termination pathways.

Abstract

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. RNA polymerases must terminate gene transcription accurately to produce diverse RNAs. Here, the authors show that human RNA polymerase III follows conserved dual termination pathways at a single terminator, preferentially releasing RNA and DNA concurrently over sequentially as intrinsic fail-safes.

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