Lanicek, M.; Sedova, K.; Hasanova, Z.; Porrua, O.; Sebesta, M.

Persistent R-loops and paused or stalled RNA polymerase II (RNAPII) elongation complexes can threaten genome stability. Senataxin (SETX) contributes to R-loop resolution and RNAPII regulation, but their intimate connection during transcription makes the individual contributions of these activities difficult to distinguish in cells. To determine whether these activities rely on the same molecular features, we investigated the structure and biochemical properties of the SETX helicase domain (SETX-HD). Cryo-EM defines the conserved helicase core and RNA-binding channel of SETX-HD, while biochemical analyses identify distinct roles for two peripheral elements: a previously uncharacterised C-terminal RNA-binding domain (CT-RBD) and the prong subdomain. In reconstituted mammalian transcription assays, SETX-HD promotes passage beyond a defined RNAPII pause and elongation-complex dissociation on the same template. The CT-RBD stabilises RNA engagement and is required for efficient R-loop unwinding, whereas its deletion preserves both RNAPII-associated activities. Conversely, replacing the native SETX prong with its fungal Sen1 counterpart preserves R-loop unwinding but impairs both RNAPII outcomes. These reciprocal separation-of-function effects establish that a common SETX-HD RNA-translocation core depends on distinct peripheral elements for R-loop unwinding and action on mammalian RNAPII. This separation provides a direct route to determine how these activities contribute to SETX function in cells.